Multifunctional degradable nanoparticles with control over size and functionalities

ABSTRACT

In one aspect, the invention relates to polymers, crosslinked polymers, functionalized polymers, nanoparticles, and functionalized nanoparticles and methods of making and using same. In one aspect, the invention relates to degradable polymers and degradable nanoparticles. In one aspect, the invention relates to methods of preparing degradable nanoparticles and, more specifically, methods of controlling particle size during the preparation of degradable nanoparticles. In one aspect, the degradable nanoparticles are useful for complexing, delivering, and releasing payloads, including pharmaceutically active payloads. This abstract is intended as a scanning tool for purposes of searching in the particular art and is not intended to be limiting of the present invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.15/049,781, filed Feb. 22, 2016; which is a Continuation of U.S. patentapplication Ser. No. 13/520,775, filed Jan. 28, 2013; which is aNational Phase Application of International Application No.PCT/US2011/020148, filed Jan. 4, 2011; which claims priority to U.S.application Ser. No. 12/651,710, filed Jan. 4, 2010; which is aContinuation-in-Part of International Application No. PCT/US2008/082529filed Nov. 5, 2008; which claims priority to U.S. ProvisionalApplication No. 61/101,039 filed Sep. 29, 2008, U.S. ProvisionalApplication No. 61/100,752 filed Sep. 28, 2008, U.S. ProvisionalApplication No. 61/038,041 filed Mar. 19, 2008, and 60/985,608, filedNov. 5, 2007, which applications are hereby incorporated herein byreference in their entireties.

ACKNOWLEDGMENT

This invention was made with government support under a CAREER AwardCHE-0645737 awarded by the National Science Foundation. The governmenthas certain rights in the invention.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted Aug. 21, 2017 as a text file named“22000_0193U4_ST25.txt,” created on Aug. 21, 2017, and having a size of1,250 bytes is hereby incorporated by reference pursuant to 37 C.F.R. §1.52(e)(5).

BACKGROUND

Biodegradable nanoparticles have received increasing attention asversatile drug delivery scaffolds to enhance the efficacy oftherapeutics. Effectiveness of delivery, however, can be influenced bythe particle size and morphology, as these parameters can greatly affectthe biological function and fate of the material. [Zweers, M. L. T.;Grijpma, D. W.; Engbers, G. H. M.; Feijen, J., J. Controlled Release2003, 87, 252-254.] Narrowly dispersed particles are highly preferredfor use in delivery or sensing applications with respect to monitoringand predicting their behavior as their exhibit a more constant responseto external stimuli. [Lubetkin, S.; Mulqueen, P.; Paterson, E. Pesti.Sci. 1999, 55, 1123-1125.]

One disadvantage of conventional methods is the irreproducibility in thesize and shape of the particles, since these can be profoundlyinfluenced by the stabilizer and the solvent used. [Kumar, M. N. V. R.;Bakowsky, U.; Lehr, C. M., Biomaterials 2004, 25, 1771-1777.] Anothermajor drawback of conventional biodegradable nanoparticles, based onpoly(ε-caprolactone) and other aliphatic polyesters, is the lack ofpendant functional groups, which can make physiochemical, mechanical,and biological properties difficult to modify. [(a) Riva, R.; Lenoir,S.; Jerome, R.; Lecomte, P. Polymer 2005, 46, 8511-8518. (b) Sasatsu,M.; Onishi, H.; Machida, Y. Inter. J. Pharm. 2006, 317, 167-174.] Theavailability of functional groups is a desirable means of tailoring theproperties of a particle, including hydrophilicity, biodegradation rate,and bioadhesion.

Therefore, there remains a need for methods and compositions thatovercome these deficiencies and that effectively provide functionalized,degradable nanoparticles with reproducibility in particle size andshape.

SUMMARY

In accordance with the purpose(s) of the invention, as embodied andbroadly described herein, the invention, in one aspect, relates topolymers, crosslinked polymers, functionalized polymers, nanoparticles,and functionalized nanoparticles and methods of making and using same.

Disclosed are methods of administering a pharmaceutical or biologicallyactive agent to a cell comprising contacting the cell with a degradablepolyester nanoparticle-agent complex (nanoparticle complex) therebyadministering the pharmaceutical or biologically active agent to thecell.

Also disclosed are methods of modulating a receptor on a cell comprisingcontacting the receptor with a degradable polyester nanoparticlepharmaceutical or biologically active agent complex, wherein one or morepharmaceutical agents is encapsulated by a degradable polyesternanoparticle.

Also disclosed are methods of inhibiting VEGF activity in an eye in asubject comprising administering to the subject a degradable polyesternanoparticle pharmaceutical or biologically active agent complex(nanoparticle complex).

Also disclosed are methods of inhibiting carboninc anhydrase activity inan eye in a subject comprising administering to the subject an effectiveamount of a degradable polyester nanoparticle pharmaceutical orbiologically active agent complex (nanoparticle complex).

Also disclosed are methods of treating a ophthalmic disorder comprisingadministering to a subject an effective amount of a degradable polyesternanoparticle pharmaceutical or biologically active agent complex(nanoparticle complex).

Also disclosed are crosslinked degradable nanoparticle having apolyester backbone and one or more crosslinks having a structureselected from:

wherein Y is O, S, or N—R, wherein R is C1-C4 alkyl;

wherein L is a divalent alkyl chain or alkyloxyalkyl chain.

Also disclosed are compositions comprising a degradable polyesternanoparticle and, encapsulated therein, a biologically active agent, apharmaceutically active agent, or an imaging agent.

Also disclosed are kits comprising a first degradable polyesternanoparticle and a first biologically active agent, firstpharmaceutically active agent, or first imaging agent encapsulatedwithin the first nanoparticle, and one or more of: a second biologicallyactive agent, second pharmaceutically active agent, or second imagingagent encapsulated within the first nanoparticle, wherein the firstbiologically active agent, first pharmaceutically active agent, or firstimaging agent is different from the second biologically active agent,second pharmaceutically active agent, or second imaging agent; or asecond degradable polyester nanoparticle and a second biologicallyactive agent, second pharmaceutically active agent, or second imagingagent encapsulated within the second nanoparticle, wherein the firstbiologically active agent, first pharmaceutically active agent, or firstimaging agent is different from the second biologically active agent,second pharmaceutically active agent, or second imaging agent; apharmaceutically acceptable carrier; or instructions for treating adisorder known to be treatable by the first biologically active agent orfirst pharmaceutically active agent.

Also disclosed are the products of the disclosed methods.

Also disclosed are methods of intracellular delivery comprisingadministering an effective amount of a disclosed nanoparticle to asubject.

Also disclosed are methods for the manufacture of a medicament fordelivery of a biologically active agent, a pharmaceutically activeagent, and/or an imaging moiety comprising combining at least onedisclosed polymer or at least one disclosed nanoparticle with apharmaceutically acceptable carrier.

Also disclosed are uses of a disclosed polymer or a disclosednanoparticle to deliver a biologically active agent, a pharmaceuticallyactive agent, and/or an imaging moiety.

Also disclosed are pharmaceutical compositions comprising atherapeutically effective amount of one or more disclosed polymer and/orone or more disclosed nanoparticle and a pharmaceutically acceptablecarrier for administration in a subject, for example, a mammal.

Also disclosed are pharmaceutical compositions for diagnosing, treating,and/or preventing ophthalmic disorders, the compositions comprising atherapeutically effective amount of one or more disclosed polymer and/orone or more disclosed nanoparticle and a pharmaceutically acceptablecarrier for administration in a subject, for example, a mammal. In oneaspect, the compositions can be administered transcomeally.

Also disclosed are microparticles, and/or larger networks, for use asmaterials for tissue engineering and biogels in biomedical devices.

While aspects of the present invention can be described and claimed in aparticular statutory class, such as the system statutory class, this isfor convenience only and one of skill in the art will understand thateach aspect of the present invention can be described and claimed in anystatutory class. Unless otherwise expressly stated, it is in no wayintended that any method or aspect set forth herein be construed asrequiring that its steps be performed in a specific order. Accordingly,where a method claim does not specifically state in the claims ordescriptions that the steps are to be limited to a specific order, it isno way intended that an order be inferred, in any respect. This holdsfor any possible non-express basis for interpretation, including mattersof logic with respect to arrangement of steps or operational flow, plainmeaning derived from grammatical organization or punctuation, or thenumber or type of aspects described in the specification.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying figures, which are incorporated in and constitute apart of this specification, illustrate several aspects and together withthe description serve to explain the principles of the invention.

FIG. 1 shows hydrolytic biodegradation studies of (▴) 725.1±94.3 nmpoly(vl-evl) nanoparticles to (♦) 30.71±2.21 nm AB nanoparticles. Allparticles are non-emulsified.

FIG. 2 shows cytotoxicity of vitamin E TPGS formulated nanoparticles onHeLa cells after 24 h incubation using the MTT assay. Fitted curve showscell viability of the HeLa cell line.

FIG. 3 shows in vitro degradation profile of vitamin E TPGS formulatedpoly(vl-evl-avl-opd) nanoparticles of 53 nm in DPBS at pH 7.4 and 37° C.over a period of 384 h (16 days).

FIG. 4 shows in vitro release profile of paclitaxel from particlesloaded with 11.3% paclitaxel prepared with the emulsification process.The drug release was performed in DPBS at pH 7.4 and 37° C. for 60 days.The cumulative release profile shows a desirable controlled andsustained release of paclitaxel from the nanoparticles.

FIG. 5 shows transmission electron microscopy (TEM) images of (A)nanoparticles without taxol with a size of 53 nm and (B) nanoparticlesencapsulated with 11.3% taxol with a size dimension of 57 nm.

FIG. 6 shows synthesis of a targeted, water-soluble nanoparticle drugdelivery system involving thiol-ene “click” chemistries and drug loadingvia developed emulsification process after post-modification.

FIG. 7 shows encapsulation of brimonidine in nanoparticles.

FIG. 8 shows drug release of disclosed nanoparticles in comparison toother polyester based nanoparticle systems.

FIG. 9 shows synthesis and validation of optimized nanoparticlesdifferentiated by size, release kinetics, incorporated drug, targetingparameter, and/or imaging modality.

FIG. 10 shows multifunctional linear polyester precursors with epoxidecross-linking entity.

FIG. 11 shows TEM images of AB nanoparticles; (1) 2 equivalents ofamine; (2) 5 equivalents of amine; (3) 8 equivalents of amine.

FIG. 12 shows polynomial increase of nanoparticle diameter (nm) withincrease of diamine cross-linker; (▪) ABD nanoparticles; (♦) ABnanoparticles; (●) ABC nanoparticles.

FIG. 13 shows ¹H NMR overlay for poly(vl-evl) nanoparticles withincreasing cross-linking.

FIG. 14 shows polynomial increase of nanoparticle diameter (nm) withincrease of diamine cross-linker for (♦) AB nanoparticles from FIG. 12.

FIG. 15 shows polynomial increase of nanoparticle diameter (nm) withincrease of diamine cross-linker for (▪) ABD nanoparticles from FIG. 12.

FIG. 16 shows polynomial increase of nanoparticle diameter (nm) withincrease of diamine cross-linker for (●) ABC nanoparticles from FIG. 12.

FIG. 17 shows polynomial increase of nanoparticle diameter (nm) withincrease of diamine cross-linker for AB nanoparticles from poly(vl-evl)(2% evl) (▪).

FIG. 18 shows polynomial increase of nanoparticle diameter (nm) withincrease of diamine cross-linker for AB nanoparticles from poly(vl-evl)(7% evl) (♦).

FIG. 19 shows polynomial increase of nanoparticle diameter (nm) withincrease of diamine cross-linker for AB nanoparticles from poly(vl-evl)(19% evl) (♦).

FIG. 20 shows a schematic representation of the structures for FD-1 andFD-2.

FIG. 21 shows time course of internalization of (a) FD-1 and (b) FD-2into NIH-3T3 Fibroblasts at 37° C. The conjugate concentration was 10μM.

FIG. 22 shows the effect of temperature on (a) FD-1 and (b) FD-2internalization. The human microvascular endothelial cells (HMEC) cellswere incubated with conjugates (10 uM) for 2.5 min at 4° C. or at 37° C.

FIG. 23 shows the effect of temperature on (a) FD-1 and (b) FD-2internalization. The HMEC cells were incubated with conjugates (1 uM)for 30 min at 4° C. or at 37° C.

FIG. 24 shows control experiments: (a) The HMEC cells were incubatedwith free FITC conjugates (10 uM) for 60 min at 37° C. (b) The HMECcells were incubated with Boc-protected guanidinylated FD-2 (10 uM) for60 min at 37° C.

FIG. 25A and FIG. 25B show an exemplary synthetic scheme for thepreparation of FD-1, FD-2, and intermediates thereof.

FIG. 26 shows a schematic of exemplary multimodal nanoparticles.

FIG. 27 shows an exemplary conjugation of a disclosed dendrimericmaterial with a disclosed cross-linked organic nanoparticle.

FIG. 28 shows a schematic illustrating a disclosed delivery system(e.g., gene delivery).

FIG. 29 illustrates preparation of a disclosed delivery system (e.g.,gene delivery).

FIG. 30 shows micrographs demonstrating mitrochondrial localization ofthe disclosed delivery systems (e.g., gene delivery).

FIG. 31 shows micrographs demonstrating uptake of a disclosed deliverysystem (e.g., gene delivery) in ciEndothelial cells.

FIG. 32 demonstrates the flexibility of assembly of the discloseddelivery systems.

FIG. 33 shows micrographs of HeLa cells exposed 10 μM FD-1 for 1 h,fixed with 3.3% paraformaldehyde, stained with 100 nM Mitotracker® Red580 FM. The illuminated regions show cell penetration (left),mitochondria location (center), and overlap (right).

FIG. 34 shows micrographs of HeLa cells exposed 20 μM FD-2 for 1 h,fixed with 3.3% paraformaldehyde, stained with 100 nM Mitotracker® Red580 FM. The illuminated regions show cell penetration (left),mitochondria location (center), and overlap (right).

FIG. 35 shows micrographs demonstrating intercellular transport of anaprotinin-fluorophore-transporter conjugate (FD-1, illustrated) intoHAEC cells.

FIG. 36 shows micrographs demonstrating intercellular transport of anaprotinin-fluorophore-transporter conjugate (FD-2) into HAEC cells.

FIG. 37 shows micrographs demonstrating no uptake (i.e., nointercellular transport into HAEC cells) of a controlaprotinin-fluorophore conjugate (illustrated).

FIG. 38 illustrates several chemical strategies for binding transportermoities to various protein functional groups (e.g., amine, thiol,carbonyl).

FIG. 39 presents strategies for vaccine development by incorporation ofaprotinin through conjugation to carbonyl-functionalized proteins (e.g.,tyrosine residues) by Mannich reaction.

FIG. 40 illustrates incorporation of fluorophores through conjugation tocarbonyl-functionalized proteins (e.g., tyrosine residues) by Mannichreaction.

FIG. 41 illustrates incorporation of transporter moieties throughconjugation to carbonyl-functionalized proteins.

FIG. 42 shows TEM analysis of the nanoparticles (225.6 nm) produced fromcrosslinking of poly(vl-evl-avl-opd) (ABbD).

FIG. 43 shows the particle size distribution measured by dynamic lightscatter analysis of “one-pot” nanoparticles (272.3±23.3 nm) producedfrom crosslinking of poly(vl-evl-avl-opd) (ABbD).

FIG. 44 shows a scheme for a thiol exchange reaction with an IgGantibody to form an IgGMT bioconjugate.

FIG. 45 shows microscopy images of uptake of IgGMT into HEp-2 cells for10 min, 30 min, 1 h, 2 h, 6 h and negative control experiment (NC) withAlexa Fluor® 568 labeled IgG.

FIG. 46 shows microscopy images of HEp-2 cells infected with RSV for 24h, washed and imaged 48 h after infection for the fluorescence of GFP(c). HEp-2 cells infected with RSV for 24 h, incubated for 30 min withIgGMT and imaged after 48 h for the fluorescence of GFP (a) and AlexaFluor® 568 of the IgGMT (b), merged images (a) and (b) (merged a+b).

FIG. 47 shows microscopy images of HEp-2 cells infected with RSV for 24h, incubated for 30 min with IgGMT and imaged immediately for the greenfluorescence of the GFP (a) and the red fluorescence of the IgGMTconjugate (b), merged images of (a) and (b) (a+b merged). HEp-2 cellsinfected with RSV for 24 h, incubated for 30 min with IgGMT and imagedafter 48 h for the fluorescence of GFP (a) and Alexa Fluor® 568 of theIgGMT conjugate (b), merged images (a) and (b) (merged a+b).

FIG. 48 shows results for a radiation guided Nanoparticle-peptidetargeting in a Lewis-Lung Carcinoma Tumor Model. The peptide shown isSeq. I.D. 1.

FIG. 49 shows a scheme for delivery of a biological active substance.The peptide shown in Seq. I.D. 1.

FIGS. 50A-D shows fluorescence microscopy images of portions of the eyeof a rat after administration of a nanoparticle bioconjugate comprisingan imaging agent.

FIG. 51 shows a schematic of nanoparticle formation from poly(vl-opd)via reductive amination.

FIG. 52 shows a transmission electron microscopy (TEM) image ofnanoparticles formed from poly(vl-opd) via reductive amination.

FIGS. 53A-F shows deposition of DiO dye on the retinal surface over timeafter a single injection of DiO nanoparticle complex.

FIGS. 54A-C shows deposition of DiO dye in ganglion cells over timeafter a single injection of DiO nanoparticle complex.

FIG. 55A-C shows “nanosponges,” which are three-dimensionalnano-networks formed from degradable materials, in particular, formed bycrosslinking degradable linear polyesters. FIG. 55A is a schematicrepresentation of a 50 nm degradable nanoparticle (nanosponge), 7%cross-linking density, loaded with 1.3% travatan, 0.38 mg/mL. FIG. 55Bis a schematic representation of a 400 nm degradable nanoparticle(nanosponge), 14% cross-linking density, loaded with 22.4% bimatoprost,3.58 mg/mL. FIG. 55C is a schematic representation of a 700 nmdegradable nanoparticle (nanosponge), 14% cross-linking density, loadedwith 29.35% bimatoprost, 4.7 mg/mL. In a separate example, a morecrystalline 700 nm degradable nanoparticle (nanosponge), 14%cross-linking density, was loaded with a 25.41% bimatoprost, 4.07 mg/mL.

FIG. 56 summarizes hypotensive drug trials with a 50 nm “nanosponge” (7%cross-linking density, 1.3% travatan, 0.38 mg/mL). The upper panel is agraph of intraocular pressure as a function of time after intravitrealadministration of the nanosponge (intravitreal travatan nanoparticles)(-●-) versus time after intravitreal administration of PBS (-∇-). Thelower panel is a graph of intraocular pressure as a function of timeafter intravitreal administration of topical travatan (-●-) versus timeafter intravitreal administration of the nanosponge (intravitrealtravatan nanoparticles) (-∇-).

FIG. 57 summarizes hypotensive drug trials with a Lumigan (BimatoprostOphthalmic)-loaded 400 nm “nanosponge” (14% cross-linking density, 22.4%bimatoprost, 3.58 mg/mL), with a 700 nm “nanosponge” (14% cross-linkingdensity, 29.35% bimatoprost, 4.7 mg/mL), and with a 700 nm “nanosponge”(14% cross-linking density, 25.41% bimatoprost, 4.07 mg/mL). The upperpanel is a graph of intraocular pressure as a function of time afterintravitreal administration of control (PBS) (-●-) versus time afterintravitreal administration of the 400 nm nanosponge (intravitrealbimatoprost nanoparticles) (—∇—). The lower panel is a graph ofintraocular pressure as a function of time after intravitrealadministration of control (PBS) (-●-) versus time after intravitrealadministration of the 700 nm nanosponge (intravitreal bimatoprostnanoparticles) (-∇-).

FIG. 58 shows the synthesis of a doxorubicin biocongujate.

FIG. 59 shows an example crosslinking reaction, and example productthereof.

FIG. 60 shows the synthesis of a SVEC linker and attachment to theparticle.

FIG. 61 shows the attachment of a peptide with an integrated thiol groupfrom a cysteine residue to a linker-modified particle.

FIG. 62 shows the attachment of Alex Fluor dye to the free amine groupsof the particle (NHS ester to amine) and quenching of the residualamines before reductive amination of amines of peptides (bioactivecompounds) to the keto groups of the particle.

FIG. 63 shows nanoparticle formation from an allyl-functionalized ABbDlinear precursor with diamines.

FIG. 64 shows nanoparticle formation from an allyl-functionalized ABbDlinear precursor with diamines, followed by attachment to anisothiocyanate.

FIG. 65 shows nanoparticle formation from an allyl-functionalized ABbDlinear precursor with diamines, thereby providing multiplyfunctionalized degradable nanoparticles.

FIG. 66 shows a strategy for attaching dendritic transporter tonanoparticle.

FIG. 67 shows sequential modification of collapsible nanoparticles.

FIG. 68 shows attachment of a targeting peptide to the SVEC system. Thepeptide shown is Seq. I.D. 1.

FIG. 69 shows attachment of a targeting peptide to a nanoparticlesystem. The peptide shown is Seq. I.D. 2.

FIG. 70 shows functionalization of organic quantum dots viaintramolecular chain collapse. The peptide shown is Seq. I.D. 1.

FIG. 71 shows deprotection of triflate with a base and attachment ofSVEC followed by the deprotection of the acylhydrazone linker. Thepeptide shown is Seq. I.D. 1.

FIG. 72 shows the attachment of DOTA.

FIG. 73 shows functionalization of a nanoparticle with a dye for imagingthe eye for testing.

FIG. 74 shows attachment of a targeting unit; also c-RGD. The peptideshown is Seq. I.D. 1.

FIG. 75 shows c-RGD.

FIG. 76 shows the synthesis of C-RGD.

FIG. 77 shows the attachment of c-RGD.

FIG. 78 shows the combination of dendritic and peptidic scaffold.

FIG. 79 shows the synthesis of NP-P-MT-dye, ABbD-NP-596-cRGD-MT (12),utilizing thiol-ene chemistry.

FIG. 80 shows the synthesis of NP-P-MT-dye conjugate, ABbD-NP-594-MTutilizing reductive amination and thiol-ene chemistry.

Additional advantages of the invention will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or can be learned by practice of the invention. Theadvantages of the invention will be realized and attained by means ofthe elements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

DESCRIPTION

The present invention can be understood more readily by reference to thefollowing detailed description of the invention and the Examplesincluded therein.

Before the present compounds, compositions, articles, systems, devices,and/or methods are disclosed and described, it is to be understood thatthey are not limited to specific synthetic methods unless otherwisespecified, or to particular reagents unless otherwise specified, as suchmay, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular aspects only andis not intended to be limiting. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, example methods andmaterials are now described.

All publications mentioned herein are incorporated herein by referenceto disclose and describe the methods and/or materials in connection withwhich the publications are cited. The publications discussed herein areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing herein is to be construed as an admissionthat the present invention is not entitled to antedate such publicationby virtue of prior invention. Further, the dates of publication providedherein can be different from the actual publication dates, which canrequire independent confirmation.

A. Definitions

As used herein, nomenclature for compounds, including organic compounds,can be given using common names, IUPAC, IUBMB, or CAS recommendationsfor nomenclature. When one or more stereochemical features are present,Cahn-Ingold-Prelog rules for stereochemistry can be employed todesignate stereochemical priority, E/Z specification, and the like. Oneof skill in the art can readily ascertain the structure of a compound ifgiven a name, either by systemic reduction of the compound structureusing naming conventions, or by commercially available software, such asCHEMDRAW™ (Cambridgesoft Corporation, U.S.A.).

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a functionalgroup,” “an alkyl,” or “a residue” includes mixtures of two or more suchfunctional groups, alkyls, or residues, and the like.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another aspect includes from the one particular value and/orto the other particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it will be understoodthat the particular value forms another aspect. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint. It is also understood that there are a number of valuesdisclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that each unit between two particularunits are also disclosed. For example, if 10 and 15 are disclosed, then11, 12, 13, and 14 are also disclosed.

References in the specification and concluding claims to parts by weightof a particular element or component in a composition denotes the weightrelationship between the element or component and any other elements orcomponents in the composition or article for which a part by weight isexpressed. Thus, in a compound containing 2 parts by weight of componentX and 5 parts by weight component Y, X and Y are present at a weightratio of 2:5, and are present in such ratio regardless of whetheradditional components are contained in the compound.

A weight percent (wt. %) of a component, unless specifically stated tothe contrary, is based on the total weight of the formulation orcomposition in which the component is included.

As used herein, the terms “optional” or “optionally” means that thesubsequently described event or circumstance can or can not occur, andthat the description includes instances where said event or circumstanceoccurs and instances where it does not.

As used herein, the term “treatment” refers to the medical management ofa patient with the intent to cure, ameliorate, stabilize, or prevent adisease, pathological condition, or disorder. This term includes activetreatment, that is, treatment directed specifically toward theimprovement of a disease, pathological condition, or disorder, and alsoincludes causal treatment, that is, treatment directed toward removal ofthe cause of the associated disease, pathological condition, ordisorder. In addition, this term includes palliative treatment, that is,treatment designed for the relief of symptoms rather than the curing ofthe disease, pathological condition, or disorder; preventativetreatment, that is, treatment directed to minimizing or partially orcompletely inhibiting the development of the associated disease,pathological condition, or disorder; and supportive treatment, that is,treatment employed to supplement another specific therapy directedtoward the improvement of the associated disease, pathologicalcondition, or disorder.

As used herein, the term “diagnosed” means having been subjected to aphysical examination by a person of skill, for example, a physician, andfound to have a condition that can be diagnosed or treated by thecompounds, compositions, or methods disclosed herein. For example,“diagnosed with an occular disorder” means having been subjected to aphysical examination by a person of skill, for example, a physician, andfound to have a disorder of the eye or eyes prior to treatment. As afurther example, “diagnosed with glaucoma” means having been subjectedto a physical examination by a person of skill, for example, aphysician, and found to have glaucoma (e.g., “open angle” or “closedangle”) prior to treatment.

As used herein, the phrase “identified to be in need of treatment for adisorder,” or the like, refers to selection of a subject based upon needfor treatment of the disorder. For example, a subject can be identifiedas having a need for treatment of a disorder (e.g., an occular disorder,glaucoma, “open angle” glaucoma, or “closed angle” glaucoma) based uponan earlier diagnosis by a person of skill and thereafter subjected totreatment for the disorder. As a further example, a subject can beidentified as having a need for treatment of a disorder afteradministration by recognition of the subject's response to the treatment(i.e., alleviation of symptoms or prevention of disorder). It iscontemplated that the identification can, in one aspect, be performed bya person different from the person making the diagnosis. It is alsocontemplated, in a further aspect, that the administration can beperformed by one who subsequently performed the administration.

As used herein, the term “prevent” or “preventing” refers to precluding,averting, obviating, forestalling, stopping, or hindering something fromhappening, especially by advance action. It is understood that wherereduce, inhibit or prevent are used herein, unless specificallyindicated otherwise, the use of the other two words is also expresslydisclosed.

As used herein, the terms “administering” and “administration” refer toany method of providing a pharmaceutical preparation to a subject. Suchmethods are well known to those skilled in the art and include, but arenot limited to, oral administration, transdermal administration,administration by inhalation, nasal administration, topicaladministration (such as, for example, eye drops, creams, salves, andirrigation), intravaginal administration, ophthalmic administration,intraaural administration, intracerebral administration, rectaladministration, and parenteral administration, including injectable suchas intravenous administration, intra-arterial administration,intramuscular administration, and subcutaneous administration.Administration can be continuous or intermittent. In various aspects, apreparation can be administered therapeutically; that is, administeredto treat an existing disease or condition. In further various aspects, apreparation can be administered prophylactically; that is, administeredfor prevention of a disease or condition. It is further contemplatedthat administration methods include parenteral methods such asintravitreal, subcutaneous, intradermal, intravenous, epicutaneous,intraocular, conjunctival, subconjuctival, intracomeal, retrobulbar, andintramuscular injections.

As used herein, the term “subject” refers to a target of administration.The subject of the herein disclosed methods can be a vertebrate, such asa mammal, a fish, a bird, a reptile, or an amphibian. Thus, the subjectof the herein disclosed methods can be a human, non-human primate,horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent.The term does not denote a particular age or sex. Thus, adult andnewborn subjects, as well as fetuses, whether male or female, areintended to be covered. A patient refers to a subject afflicted with adisease or disorder. The term “patient” includes human and veterinarysubjects.

As used herein, the terms “effective amount” and “amount effective”refer to an amount that is sufficient to achieve a desired result or tohave an effect on undesired symptoms, but is generally insufficient tocause adverse side affects. The specific effective dose level for anyparticular patient will depend upon a variety of factors including thedisorder being treated and the severity of the disorder; the specificcomposition employed; the age, body weight, general health, sex and dietof the patient; the time of administration; the route of administration;the rate of excretion of the specific compound employed; the duration ofthe treatment; drugs used in combination or coincidental with thespecific compound employed and like factors well known in the medicalarts. For example, it is well within the skill of the art to start dosesof a compound at levels lower than those required to achieve the desiredeffect and to gradually increase the dosage until the desired effect isachieved. If desired, the effective daily dose can be divided intomultiple doses for purposes of administration. Consequently, single dosecompositions can contain such amounts or submultiples thereof to make upthe daily dose. The dosage can be adjusted by the individual physicianin the event of any contraindications. Dosage can vary, and can beadministered in one or more dose administrations daily, for one orseveral days. Guidance can be found in the literature for appropriatedosages for given classes of pharmaceutical products. In a furtheraspect, a preparation can be administered in a “diagnostically effectiveamount”; that is, an amount effective for diagnosis of a disease orcondition. In a further aspect, a preparation can be administered in a“therapeutically effective amount”; that is, an amount effective fortreatment of a disease or condition. In a further aspect, a preparationcan be administered in a “prophylactically effective amount”; that is,an amount effective for prevention of a disease or condition.

As used herein, the term “pharmaceutically acceptable carrier” refers tosterile aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, as well as sterile powders for reconstitution into sterileinjectable solutions or dispersions just prior to use. Examples ofsuitable aqueous and nonaqueous carriers, diluents, solvents or vehiclesinclude water, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol and the like), carboxymethylcellulose and suitablemixtures thereof, vegetable oils (such as olive oil) and injectableorganic esters such as ethyl oleate. Proper fluidity can be maintained,for example, by the use of coating materials such as lecithin, by themaintenance of the required particle size in the case of dispersions andby the use of surfactants. These compositions can also contain adjuvantssuch as preservatives, wetting agents, emulsifying agents and dispersingagents. Prevention of the action of microorganisms can be ensured by theinclusion of various antibacterial and antifungal agents such asparaben, chlorobutanol, phenol, sorbic acid and the like. It can also bedesirable to include isotonic agents such as sugars, sodium chloride andthe like. Prolonged absorption of the injectable pharmaceutical form canbe brought about by the inclusion of agents, such as aluminummonostearate and gelatin, which delay absorption. Injectable depot formsare made by forming microencapsule matrices of the drug in biodegradablepolymers such as polylactide-polyglycolide, poly(orthoesters) andpoly(anhydrides). Depending upon the ratio of drug to polymer and thenature of the particular polymer employed, the rate of drug release canbe controlled. Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissues. The injectable formulations can be sterilized, forexample, by filtration through a bacterial-retaining filter or byincorporating sterilizing agents in the form of sterile solidcompositions which can be dissolved or dispersed in sterile water orother sterile injectable media just prior to use. Suitable inertcarriers can include sugars such as lactose. Desirably, at least 95% byweight of the particles of the active ingredient have an effectiveparticle size in the range of 0.01 to 10 micrometers.

As used herein, the term “biologically active agent” or “bioactiveagent” means an agent that is capable of providing a local or systemicbiological, physiological, or therapeutic effect in the biologicalsystem to which it is applied. For example, the bioactive agent can actto control infection or inflammation, enhance cell growth and tissueregeneration, control tumor growth, act as an analgesic, promoteanti-cell attachment, and enhance bone growth, among other functions.Other suitable bioactive agents can include anti-viral agents, vaccines,hormones, antibodies (including active antibody fragments sFv, Fv, andFab fragments), aptamers, peptide mimetics, functional nucleic acids,therapeutic proteins, peptides, or nucleic acids. Other bioactive agentsinclude prodrugs, which are agents that are not biologically active whenadministered but, upon administration to a subject are converted tobioactive agents through metabolism or some other mechanism.Additionally, any of the compositions of the invention can containcombinations of two or more bioactive agents. It is understood that abiologically active agent can be used in connection with administrationto various subjects, for example, to humans (i.e., medicaladministration) or to animals (i.e., veterinary administration).

As used herein, the term “pharmaceutically active agent” includes a“drug” or a “vaccine” and means a molecule, group of molecules, complexor substance administered to an organism for diagnostic, therapeutic,preventative medical, or veterinary purposes. This term includeexternally and internally administered topical, localized and systemichuman and animal pharmaceuticals, treatments, remedies, nutraceuticals,cosmeceuticals, biologicals, devices, diagnostics and contraceptives,including preparations useful in clinical and veterinary screening,prevention, prophylaxis, healing, wellness, detection, imaging,diagnosis, therapy, surgery, monitoring, cosmetics, prosthetics,forensics and the like. This term may also be used in reference toagriceutical, workplace, military, industrial and environmentaltherapeutics or remedies comprising selected molecules or selectednucleic acid sequences capable of recognizing cellular receptors,membrane receptors, hormone receptors, therapeutic receptors, microbes,viruses or selected targets comprising or capable of contacting plants,animals and/or humans. This term can also specifically include nucleicacids and compounds comprising nucleic acids that produce a bioactiveeffect, for example deoxyribonucleic acid (DNA) or ribonucleic acid(RNA). Pharmaceutically active agents include the herein disclosedcategories and specific examples. It is not intended that the categorybe limited by the specific examples. Those of ordinary skill in the artwill recognize also numerous other compounds that fall within thecategories and that are useful according to the invention. Examplesinclude a radiosensitizer, the combination of a radiosensitizer and achemotherapeutic, a steroid, a xanthine, a beta-2-agonistbronchodilator, an anti-inflammatory agent, an analgesic agent, acalcium antagonist, an angiotensin-converting enzyme inhibitors, abeta-blocker, a centrally active alpha-agonist, an alpha-1-antagonist,carbonic anhydrase inhibitors, prostaglandin analogs, a combination ofan alpha agonist and a beta blocker, a combination of a carbonicanhydrase inhibitor and a beta blocker, an anticholinergic/antispasmodicagent, a vasopressin analogue, an antiarrhythmic agent, anantiparkinsonian agent, an antiangina/antihypertensive agent, ananticoagulant agent, an antiplatelet agent, a sedative, an ansiolyticagent, a peptidic agent, a biopolymeric agent, an antineoplastic agent,a laxative, an antidiarrheal agent, an antimicrobial agent, anantifungal agent, or a vaccine. In a further aspect, thepharmaceutically active agent can be coumarin, albumin, bromolidine,steroids such as betamethasone, dexamethasone, methylprednisolone,prednisolone, prednisone, triamcinolone, budesonide, hydrocortisone, andpharmaceutically acceptable hydrocortisone derivatives; xanthines suchas theophylline and doxophylline; beta-2-agonist bronchodilators such assalbutamol, fenterol, clenbuterol, bambuterol, salmeterol, fenoterol;antiinflammatory agents, including antiasthmatic anti-inflammatoryagents, antiarthritis antiinflammatory agents, and non-steroidalantiinflammatory agents, examples of which include but are not limitedto sulfides, mesalamine, budesonide, salazopyrin, diclofenac,pharmaceutically acceptable diclofenac salts, nimesulide, naproxene,acetominophen, ibuprofen, ketoprofen and piroxicam; analgesic agentssuch as salicylates; calcium channel blockers such as nifedipine,amlodipine, and nicardipine; angiotensin-converting enzyme inhibitorssuch as captopril, benazepril hydrochloride, fosinopril sodium,trandolapril, ramipril, lisinopril, enalapril, quinapril hydrochloride,and moexipril hydrochloride; beta-blockers (i.e., beta adrenergicblocking agents) such as sotalol hydrochloride, timolol maleate, timolhemihydrate, levobunolol hydrochloride, esmolol hydrochloride,carteolol, propanolol hydrochloride, betaxolol hydrochloride, penbutololsulfate, metoprolol tartrate, metoprolol succinate, acebutololhydrochloride, atenolol, pindolol, and bisoprolol fumarate; centrallyactive alpha-2-agonists (i.e., alpha adrenergic receptor agonist) suchas clonidine, brimonidine tartrate, and apraclonidine hyrochloride;alpha-1-antagonists such as doxazosin and prazosin;anticholinergic/antispasmodic agents such as dicyclomine hydrochloride,scopolamine hydrobromide, glycopyrrolate, clidinium bromide, flavoxate,and oxybutynin; vasopressin analogues such as vasopressin anddesmopressin; prostaglandin analogs such as latanoprost, travoprost, andbimatoprost; cholinergics (i.e., acetylcholine receptor agonists) suchas pilocarpine hydrochloride and carbachol; glutamate receptor agonistssuch as the N-methyl D-aspartate receptor agonist memantine;anti-Vascular endothelial growth factor (VEGF) aptamers such aspegaptanib; anti-VEGF antibodies (including but not limited toanti-VEGF-A antibodies) such as ranibizumab and becacizumab; carbonicanhydrase inhibitors such as methazolamide, brinzolamide, dorzolamidehydrochloride, and acetazolamide; antiarrhythmic agents such asquinidine, lidocaine, tocainide hydrochloride, mexiletine hydrochloride,digoxin, verapamil hydrochloride, propafenone hydrochloride, flecainideacetate, procainamide hydrochloride, moricizine hydrochloride, anddisopyramide phosphate; antiparkinsonian agents, such as dopamine,L-Dopa/Carbidopa, selegiline, dihydroergocryptine, pergolide, lisuride,apomorphine, and bromocryptine; antiangina agents and antihypertensiveagents such as isosorbide mononitrate, isosorbide dinitrate,propranolol, atenolol and verapamil; anticoagulant and antiplateletagents such as coumadin, warfarin, acetylsalicylic acid, andticlopidine; sedatives such as benzodiazapines and barbiturates;ansiolytic agents such as lorazepam, bromazepam, and diazepam; peptidicand biopolymeric agents such as calcitonin, leuprolide and other LHRHagonists, hirudin, cyclosporin, insulin, somatostatin, protirelin,interferon, desmopressin, somatotropin, thymopentin, pidotimod,erythropoietin, interleukins, melatonin, granulocyte/macrophage-CSF, andheparin; antineoplastic agents such as etoposide, etoposide phosphate,cyclophosphamide, methotrexate, 5-fluorouracil, vincristine,doxorubicin, cisplatin, hydroxyurea, leucovorin calcium, tamoxifen,flutamide, asparaginase, altretamine, mitotane, and procarbazinehydrochloride; laxatives such as senna concentrate, casanthranol,bisacodyl, and sodium picosulphate; antidiarrheal agents such asdifenoxine hydrochloride, loperamide hydrochloride, furazolidone,diphenoxylate hdyrochloride, and microorganisms; vaccines such asbacterial and viral vaccines; antimicrobial agents such as penicillins,cephalosporins, and macrolides, antifungal agents such as imidazolic andtriazolic derivatives; and nucleic acids such as DNA sequences encodingfor biological proteins, and antisense oligonucleotides. It isunderstood that a pharmaceutically active agent can be used inconnection with administration to various subjects, for example, tohumans (i.e., medical administration) or to animals (i.e., veterinaryadministration).

As used herein, the term “ophthalmic disorders” and/or “ophthalmicconditions” refers to ophthalmic diseases, conditions, and/or disordersincluding, without limitation, those associated with the anteriorchamber of the eye (i.e., hyphema, synechia); the choroid (i.e.,choroidal detachment, choroidal melanoma, multifocal choroidopathysyndromes); the conjunctiva (i.e., conjunctivitis, cicatricialpemphigoid, filtering Bleb complications, conjunctival melanoma,Pharyngoconjunctival Fever, pterygium, conjunctival squamous cellcarcinoma); connective tissue disorders (i.e., ankylosing spondylitis,pseudoxanthoma elasticum, corneal abrasion or edema, limbal dermoid,crystalline dystrophy keratits, keratoconjunctivitis, keratoconus,keratopathy (including but not limited to Thygeson's superficialpunctuate keratopathy), megalocomea, corneal ulcer); dermatologicdisorders (i.e., ecrodermatitis enteropathica, atopic dermatitis, ocularrosacea, psoriasis, Stevens-Johnson syndrome); endocrine disorders(i.e., pituitary apoplexy); extraocular disorders (i.e., Abducens NervePalsy, Brown syndrome, Duane syndrome, esotropia, exotropia, oculomotornerve palsy); genetic disorders (i.e., albinism, Down syndrome, PetersAnomaly); the globe (i.e., anophthalmos, endophthalmitis); hematologicand cardiovascular disorders (i.e., Giant Cell Arteritis, hypertension,leukemias, Ocular Ischemic syndrome, sickle cell disease); infectiousdiseases (i.e., actinomycosis, botulism, HIV, diphtheria, Escherichiacoli, Tuberculosis, ocular manifestations of syphilis); intraocularpressure (i.e., glaucoma, ocular hypotony, Posner-Schlossman syndrome),the iris and ciliary body (i.e., aniridia, iris prolaps, juvenilexanthogranuloma, ciliary body melanoma, iris melanoma, uveitis); thelacrimal system (i.e., alacrima, Dry Eye syndrome, lacrimal glandtumors); the lens (i.e., cataract, ectopia lentis, intraocular lensdecentration or dislocation); the lid (i.e., blepharitis,dermatochalasis, distichiasis, ectropion, eyelid coloboma, Floppy Eyesyndrome, trichiasis, xanthelasma); metabolic disorders (i.e., gout,hyperlipoproteinemia, Oculocerebrorenal syndrome); neurologic disorders(i.e., Bell Palsy, diplopia, multiple sclerosis); general ophthalmologic(i.e., red eye, cataracts, macular degeneration, red eye, maculardegeneration); the optic nerve (i.e., miningioma, optic neuritis, opticneuropathy, papilledema); the orbit (i.e., orbital cellulits, orbitaldermoid, orbital tumors); phakomatoses (i.e., ataxia-telangiectasia,neurofibromatosis-1); presbyopia; the pupil (i.e., anisocoria, Homersyndrome); refractive disorders (i.e., astigmatism, hyperopia, myopia);the retina (i.e., Coats disease, Eales disease, macular edema,retinitis, retinopathy); and the sclera (i.e., episcleritis, scleritis).

As used herein, the terms “imaging moiety” and “imaging agent” refer toany chemical groups or substance useful for imaging applications, asknown to those of skill in the art. Examples of imaging agents includeradioconjugate, cytotoxin, cytokine, Gadolinium-DTPA or a quantum dot,iron oxide, manganese oxide, and fluorescent agents such as Alexa Fluordyes and Neuro DiO. In one aspect, an imaging agent can be provided innanoparticular form or in microparticular form. In a further aspect, animaging agent comprises Gadolinium-DTPA and iron oxide nanoparticles(magnetite), as specific MRI contrast agents. In a yet further aspect,an imaging agent comprises at least one near infrared dye, for examplenear infrared dyes based on a porphyrin and/or a phthalocyanine. SeeGhoroghchian et al., Near-infrared-emissive polymersomes: Self-assembledsoft matter for in vivo optical imaging, PNAS, 2005, vol. 102, no. 8,2922-2927.

As used herein, the term “polymer” refers to a relatively high molecularweight organic compound, natural or synthetic, whose structure can berepresented by a repeated small unit, the monomer (e.g., polyethylene,rubber, cellulose). Synthetic polymers are typically formed by additionor condensation polymerization of monomers.

As used herein, the term “copolymer” refers to a polymer formed from twoor more different repeating units (monomer residues). By way of exampleand without limitation, a copolymer can be an alternating copolymer, arandom copolymer, a block copolymer, or a graft copolymer. It is alsocontemplated that, in certain aspects, various block segments of a blockcopolymer can themselves comprise copolymers.

As used herein, the term “oligomer” refers to a relatively low molecularweight polymer in which the number of repeating units is between two andten, for example, from two to eight, from two to six, or form two tofour. In one aspect, a collection of oligomers can have an averagenumber of repeating units of from about two to about ten, for example,from about two to about eight, from about two to about six, or formabout two to about four.

As used herein, the term “reactive residue” refers to a moiety (e.g., amonomer residue) capable of undergoing chemical reaction at a reactiontemperature and/or in response to a stimulus to form a reactiveintermediate. In one aspect, a reactive residue is a moiety capableundergoing an intramolecular cross-linking reaction to provideintramolecular chain collapse.

As used herein, the term “polymerizable group” refers to a group (i.e.,a chemical functionality) capable of undergoing a polymerizationreaction at a polymerization temperature and/or in response to apolymerization initiator to form a polymer or an oligomer. In oneaspect, the polymerization reaction is a radical polymerization (e.g., avinyl polymerization). It is understood that catalysts can be employedin connection with the polymerization reaction. It is contemplated that,in various aspects, polymerizable groups can be used in step-growth orchain growth reactions. Exemplary polymerizable groups include residuesof vinyl, styryl, acryloyl, methacryloyl, aryl, and heteroarylcompounds.

A residue of a chemical species, as used in the specification andconcluding claims, refers to the moiety that is the resulting product ofthe chemical species in a particular reaction scheme or subsequentformulation or chemical product, regardless of whether the moiety isactually obtained from the chemical species. Thus, an ethylene glycolresidue in a polyester refers to one or more —OCH₂CH₂O— units in thepolyester, regardless of whether ethylene glycol was used to prepare thepolyester. Similarly, a sebacic acid residue in a polyester refers toone or more —CO(CH₂)₈CO— moieties in the polyester, regardless ofwhether the residue is obtained by reacting sebacic acid or an esterthereof to obtain the polyester.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, and aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described below. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this disclosure, the heteroatoms, such as nitrogen, canhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms. This disclosure is not intended to be limited in any mannerby the permissible substituents of organic compounds. Also, the terms“substitution” or “substituted with” include the implicit proviso thatsuch substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., a compound that does not spontaneouslyundergo transformation such as by rearrangement, cyclization,elimination, etc.

In defining various terms, “A¹,” “A²,” “A³,” and “A⁴” are used herein asgeneric symbols to represent various specific substituents. Thesesymbols can be any substituent, not limited to those disclosed herein,and when they are defined to be certain substituents in one instance,they can, in another instance, be defined as some other substituents.

The term “alkyl” as used herein is a branched or unbranched saturatedhydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl,isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. The alkylgroup can also be substituted or unsubstituted. The alkyl group can besubstituted with one or more groups including, but not limited to,optionally substituted alkyl, cycloalkyl, alkoxy, amino, ether, halide,hydroxy, nitro, silyl, sulfo-oxo, or thiol, as described herein. A“lower alkyl” group is an alkyl group containing from one to six carbonatoms.

Throughout the specification “alkyl” is generally used to refer to bothunsubstituted alkyl groups and substituted alkyl groups; however,substituted alkyl groups are also specifically referred to herein byidentifying the specific substituent(s) on the alkyl group. For example,the term “halogenated alkyl” specifically refers to an alkyl group thatis substituted with one or more halide, e.g., fluorine, chlorine,bromine, or iodine. The term “alkoxyalkyl” specifically refers to analkyl group that is substituted with one or more alkoxy groups, asdescribed below. The term “alkylamino” specifically refers to an alkylgroup that is substituted with one or more amino groups, as describedbelow, and the like. When “alkyl” is used in one instance and a specificterm such as “alkylalcohol” is used in another, it is not meant to implythat the term “alkyl” does not also refer to specific terms such as“alkylalcohol” and the like.

This practice is also used for other groups described herein. That is,while a term such as “cycloalkyl” refers to both unsubstituted andsubstituted cycloalkyl moieties, the substituted moieties can, inaddition, be specifically identified herein; for example, a particularsubstituted cycloalkyl can be referred to as, e.g., an“alkylcycloalkyl.” Similarly, a substituted alkoxy can be specificallyreferred to as, e.g., a “halogenated alkoxy,” a particular substitutedalkenyl can be, e.g., an “alkenylalcohol,” and the like. Again, thepractice of using a general term, such as “cycloalkyl,” and a specificterm, such as “alkylcycloalkyl,” is not meant to imply that the generalterm does not also include the specific term.

The term “cycloalkyl” as used herein is a non-aromatic carbon-based ringcomposed of at least three carbon atoms. Examples of cycloalkyl groupsinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, norbornyl, and the like. The term “heterocycloalkyl” is atype of cycloalkyl group as defined above, and is included within themeaning of the term “cycloalkyl,” where at least one of the carbon atomsof the ring is replaced with a heteroatom such as, but not limited to,nitrogen, oxygen, sulfur, or phosphorus. The cycloalkyl group andheterocycloalkyl group can be substituted or unsubstituted. Thecycloalkyl group and heterocycloalkyl group can be substituted with oneor more groups including, but not limited to, optionally substitutedalkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl,sulfo-oxo, or thiol as described herein.

The term “polyalkylene group” as used herein is a group having two ormore CH₂ groups linked to one another. The polyalkylene group can berepresented by the formula —(CH₂)_(a)—, where “a” is an integer of from2 to 500.

The terms “alkoxy” and “alkoxyl” as used herein to refer to an alkyl orcycloalkyl group bonded through an ether linkage; that is, an “alkoxy”group can be defined as —OA¹ where A¹ is alkyl or cycloalkyl as definedabove. “Alkoxy” also includes polymers of alkoxy groups as justdescribed; that is, an alkoxy can be a polyether such as —OA¹-OA² or—OA¹-(OA²)_(a)-OA³, where “a” is an integer of from 1 to 200 and A¹, A²,and A³ are alkyl and/or cycloalkyl groups.

The term “alkenyl” as used herein is a hydrocarbon group of from 2 to 24carbon atoms with a structural formula containing at least onecarbon-carbon double bond. Asymmetric structures such as (A¹A²)C═C(A³A⁴)are intended to include both the E and Z isomers. This can be presumedin structural formulae herein wherein an asymmetric alkene is present,or it can be explicitly indicated by the bond symbol C═C. The alkenylgroup can be substituted with one or more groups including, but notlimited to, optionally substituted alkyl, cycloalkyl, alkoxy, alkenyl,cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino,carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro,silyl, sulfo-oxo, or thiol, as described herein.

The term “cycloalkenyl” as used herein is a non-aromatic carbon-basedring composed of at least three carbon atoms and containing at least onecarbon-carbon double bound, i.e., C═C. Examples of cycloalkenyl groupsinclude, but are not limited to, cyclopropenyl, cyclobutenyl,cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl,norbornenyl, and the like. The term “heterocycloalkenyl” is a type ofcycloalkenyl group as defined above, and is included within the meaningof the term “cycloalkenyl,” where at least one of the carbon atoms ofthe ring is replaced with a heteroatom such as, but not limited to,nitrogen, oxygen, sulfur, or phosphorus. The cycloalkenyl group andheterocycloalkenyl group can be substituted or unsubstituted. Thecycloalkenyl group and heterocycloalkenyl group can be substituted withone or more groups including, but not limited to, optionally substitutedalkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl,aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether,halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol asdescribed herein.

The term “alkynyl” as used herein is a hydrocarbon group of 2 to 24carbon atoms with a structural formula containing at least onecarbon-carbon triple bond. The alkynyl group can be unsubstituted orsubstituted with one or more groups including, but not limited to,optionally substituted alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl,alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylicacid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl,sulfo-oxo, or thiol, as described herein.

The term “cycloalkynyl” as used herein is a non-aromatic carbon-basedring composed of at least seven carbon atoms and containing at least onecarbon-carbon triple bound. Examples of cycloalkynyl groups include, butare not limited to, cycloheptynyl, cyclooctynyl, cyclononynyl, and thelike. The term “heterocycloalkynyl” is a type of cycloalkenyl group asdefined above, and is included within the meaning of the term“cycloalkynyl,” where at least one of the carbon atoms of the ring isreplaced with a heteroatom such as, but not limited to, nitrogen,oxygen, sulfur, or phosphorus. The cycloalkynyl group andheterocycloalkynyl group can be substituted or unsubstituted. Thecycloalkynyl group and heterocycloalkynyl group can be substituted withone or more groups including, but not limited to, optionally substitutedalkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl,aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether,halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol asdescribed herein.

The term “aryl” as used herein is a group that contains any carbon-basedaromatic group including, but not limited to, benzene, naphthalene,phenyl, biphenyl, phenoxybenzene, and the like. The term “aryl” alsoincludes “heteroaryl,” which is defined as a group that contains anaromatic group that has at least one heteroatom incorporated within thering of the aromatic group. Examples of heteroatoms include, but are notlimited to, nitrogen, oxygen, sulfur, and phosphorus. Likewise, the term“non-heteroaryl,” which is also included in the term “aryl,” defines agroup that contains an aromatic group that does not contain aheteroatom. The aryl group can be substituted or unsubstituted. The arylgroup can be substituted with one or more groups including, but notlimited to, optionally substituted alkyl, cycloalkyl, alkoxy, alkenyl,cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino,carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro,silyl, sulfo-oxo, or thiol as described herein. The term “biaryl” is aspecific type of aryl group and is included in the definition of “aryl.”Biaryl refers to two aryl groups that are bound together via a fusedring structure, as in naphthalene, or are attached via one or morecarbon-carbon bonds, as in biphenyl.

The term “aldehyde” as used herein is represented by the formula —C(O)H.Throughout this specification “C(O)” is a short hand notation for acarbonyl group, i.e., C═O.

The terms “amine” or “amino” as used herein are represented by theformula NA¹A²A³, where A¹, A², and A³ can be, independently, hydrogen oroptionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl,alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.

The term “carboxylic acid” as used herein is represented by the formula—C(O)OH.

The term “ester” as used herein is represented by the formula —OC(O)A¹or —C(O)OA, where A¹ can be an optionally substituted alkyl, cycloalkyl,alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl groupas described herein. The term “polyester” as used herein is representedby the formula -(A¹O(O)C-A²-C(O)O)_(a)— or -(A¹O(O)C-A²-OC(O))_(a)—,where A¹ and A² can be, independently, an optionally substituted alkyl,cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, orheteroaryl group described herein and “a” is an integer from 1 to 500.“Polyester” is as the term used to describe a group that is produced bythe reaction between a compound having at least two carboxylic acidgroups with a compound having at least two hydroxyl groups.

The term “ether” as used herein is represented by the formula A¹OA²,where A¹ and A² can be, independently, an optionally substituted alkyl,cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, orheteroaryl group described herein. The term “polyether” as used hereinis represented by the formula -(A¹O-A²O)_(a)—, where A¹ and A² can be,independently, an optionally substituted alkyl, cycloalkyl, alkenyl,cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group describedherein and “a” is an integer of from 1 to 500. Examples of polyethergroups include polyethylene oxide, polypropylene oxide, and polybutyleneoxide.

The term “halide” as used herein refers to the halogens fluorine,chlorine, bromine, and iodine.

The term “heterocycle,” as used herein refers to single and multi-cyclicaromatic or non-aromatic ring systems in which at least one of the ringmembers is other than carbon. Heterocycle includes pyridinde,pyrimidine, furan, thiophene, pyrrole, isoxazole, isothiazole, pyrazole,oxazole, thiazole, imidazole, oxazole, including, 1,2,3-oxadiazole,1,2,5-oxadiazole and 1,3,4-oxadiazole, thiadiazole, including,1,2,3-thiadiazole, 1,2,5-thiadiazole, and 1,3,4-thiadiazole, triazole,including, 1,2,3-triazole, 1,3,4-triazole, tetrazole, including1,2,3,4-tetrazole and 1,2,4,5-tetrazole, pyridine, pyridazine,pyrimidine, pyrazine, triazine, including 1,2,4-triazine and1,3,5-triazine, tetrazine, including 1,2,4,5-tetrazine, pyrrolidine,piperidine, piperazine, morpholine, azetidine, tetrahydropyran,tetrahydrofuran, dioxane, and the like.

The term “hydroxyl” as used herein is represented by the formula —OH.

The term “ketone” as used herein is represented by the formula A¹C(O)A²,where A¹ and A² can be, independently, an optionally substituted alkyl,cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, orheteroaryl group as described herein.

The term “azide” as used herein is represented by the formula —N₃.

The term “nitro” as used herein is represented by the formula —NO₂.

The term “nitrile” as used herein is represented by the formula —CN.

The term “silyl” as used herein is represented by the formula —SiAA²A³,where A¹, A², and A³ can be, independently, hydrogen or an optionallysubstituted alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl,cycloalkynyl, aryl, or heteroaryl group as described herein.

The term “sulfo-oxo” as used herein is represented by the formulas—S(O)A¹, —S(O)₂A¹, —OS(O)₂A¹, or —OS(O)₂OA¹, where A¹ can be hydrogen oran optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl,alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.Throughout this specification “S(O)” is a short hand notation for S═O.The term “sulfonyl” is used herein to refer to the sulfo-oxo grouprepresented by the formula —S(O)₂A¹, where A¹ can be hydrogen or anoptionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl,alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.The term “sulfone” as used herein is represented by the formulaA¹S(O)₂A², where A¹ and A² can be, independently, an optionallysubstituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl,cycloalkynyl, aryl, or heteroaryl group as described herein. The term“sulfoxide” as used herein is represented by the formula A¹S(O)A², whereA¹ and A² can be, independently, an optionally substituted alkyl,cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, orheteroaryl group as described herein.

The term “thiol” as used herein is represented by the formula —SH.

The term “organic residue” defines a carbon containing residue, i.e., aresidue comprising at least one carbon atom, and includes but is notlimited to the carbon-containing groups, residues, or radicals definedhereinabove. Organic residues can contain various heteroatoms, or bebonded to another molecule through a heteroatom, including oxygen,nitrogen, sulfur, phosphorus, or the like. Examples of organic residuesinclude but are not limited alkyl or substituted alkyls, alkoxy orsubstituted alkoxy, mono or di-substituted amino, amide groups, etc.Organic residues can preferably comprise 1 to 18 carbon atoms, 1 to 15,carbon atoms, 1 to 12 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbonatoms, or 1 to 4 carbon atoms. In a further aspect, an organic residuecan comprise 2 to 18 carbon atoms, 2 to 15, carbon atoms, 2 to 12 carbonatoms, 2 to 8 carbon atoms, 2 to 4 carbon atoms, or 2 to 4 carbon atoms

A very close synonym of the term “residue” is the term “radical,” whichas used in the specification and concluding claims, refers to afragment, group, or substructure of a molecule described herein,regardless of how the molecule is prepared. For example, a2,4-thiazolidinedione radical in a particular compound has the structure

regardless of whether thiazolidinedione is used to prepare the compound.In some embodiments the radical (for example an alkyl) can be furthermodified (i.e., substituted alkyl) by having bonded thereto one or more“substituent radicals.” The number of atoms in a given radical is notcritical to the present invention unless it is indicated to the contraryelsewhere herein.

“Organic radicals,” as the term is defined and used herein, contain oneor more carbon atoms. An organic radical can have, for example, 1-26carbon atoms, 1-18 carbon atoms, 1-12 carbon atoms, 1-8 carbon atoms,1-6 carbon atoms, or 1-4 carbon atoms. In a further aspect, an organicradical can have 2-26 carbon atoms, 2-18 carbon atoms, 2-12 carbonatoms, 2-8 carbon atoms, 2-6 carbon atoms, or 2-4 carbon atoms. Organicradicals often have hydrogen bound to at least some of the carbon atomsof the organic radical. One example, of an organic radical thatcomprises no inorganic atoms is a 5, 6, 7, 8-tetrahydro-2-naphthylradical. In some embodiments, an organic radical can contain 1-10inorganic heteroatoms bound thereto or therein, including halogens,oxygen, sulfur, nitrogen, phosphorus, and the like. Examples of organicradicals include but are not limited to an alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, mono-substituted amino,di-substituted amino, acyloxy, cyano, carboxy, carboalkoxy,alkylcarboxamide, substituted alkylcarboxamide, dialkylcarboxamide,substituted dialkylcarboxamide, alkylsulfonyl, alkylsulfinyl, thioalkyl,thiohaloalkyl, alkoxy, substituted alkoxy, haloalkyl, haloalkoxy, aryl,substituted aryl, heteroaryl, heterocyclic, or substituted heterocyclicradicals, wherein the terms are defined elsewhere herein. A fewnon-limiting examples of organic radicals that include heteroatomsinclude alkoxy radicals, trifluoromethoxy radicals, acetoxy radicals,dimethylamino radicals and the like.

“Inorganic radicals,” as the term is defined and used herein, contain nocarbon atoms and therefore comprise only atoms other than carbon.Inorganic radicals comprise bonded combinations of atoms selected fromhydrogen, nitrogen, oxygen, silicon, phosphorus, sulfur, selenium, andhalogens such as fluorine, chlorine, bromine, and iodine, which can bepresent individually or bonded together in their chemically stablecombinations. Inorganic radicals have 10 or fewer, or preferably one tosix or one to four inorganic atoms as listed above bonded together.Examples of inorganic radicals include, but not limited to, amino,hydroxy, halogens, nitro, thiol, sulfate, phosphate, and like commonlyknown inorganic radicals. The inorganic radicals do not have bondedtherein the metallic elements of the periodic table (such as the alkalimetals, alkaline earth metals, transition metals, lanthanide metals, oractinide metals), although such metal ions can sometimes serve as apharmaceutically acceptable cation for anionic inorganic radicals suchas a sulfate, phosphate, or like anionic inorganic radical. Inorganicradicals do not comprise metalloids elements such as boron, aluminum,gallium, germanium, arsenic, tin, lead, or tellurium, or the noble gaselements, unless otherwise specifically indicated elsewhere herein.

Compounds described herein can contain one or more double bonds and,thus, potentially give rise to cis/trans (E/Z) isomers, as well as otherconformational isomers. Unless stated to the contrary, the inventionincludes all such possible isomers, as well as mixtures of such isomers.

Unless stated to the contrary, a formula with chemical bonds shown onlyas solid lines and not as wedges or dashed lines contemplates eachpossible isomer, e.g., each enantiomer and diastereomer, and a mixtureof isomers, such as a racemic or scalemic mixture. Compounds describedherein can contain one or more asymmetric centers and, thus, potentiallygive rise to diastereomers and optical isomers. Unless stated to thecontrary, the present invention includes all such possible diastereomersas well as their racemic mixtures, their substantially pure resolvedenantiomers, all possible geometric isomers, and pharmaceuticallyacceptable salts thereof. Mixtures of stereoisomers, as well as isolatedspecific stereoisomers, are also included. During the course of thesynthetic procedures used to prepare such compounds, or in usingracemization or epimerization procedures known to those skilled in theart, the products of such procedures can be a mixture of stereoisomers.

Disclosed are the components to be used to prepare the compositions ofthe invention as well as the compositions themselves to be used withinthe methods disclosed herein. These and other materials are disclosedherein, and it is understood that when combinations, subsets,interactions, groups, etc. of these materials are disclosed that whilespecific reference of each various individual and collectivecombinations and permutation of these compounds can not be explicitlydisclosed, each is specifically contemplated and described herein. Forexample, if a particular compound is disclosed and discussed and anumber of modifications that can be made to a number of moleculesincluding the compounds are discussed, specifically contemplated is eachand every combination and permutation of the compound and themodifications that are possible unless specifically indicated to thecontrary. Thus, if a class of molecules A, B, and C are disclosed aswell as a class of molecules D, E, and F and an example of a combinationmolecule, A-D is disclosed, then even if each is not individuallyrecited each is individually and collectively contemplated meaningcombinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considereddisclosed. Likewise, any subset or combination of these is alsodisclosed. Thus, for example, the sub-group of A-E, B-F, and C-E wouldbe considered disclosed. This concept applies to all aspects of thisapplication including, but not limited to, steps in methods of makingand using the compositions of the invention. Thus, if there are avariety of additional steps that can be performed it is understood thateach of these additional steps can be performed with any specificembodiment or combination of embodiments of the methods of theinvention.

It is understood that the compositions disclosed herein have certainfunctions. Disclosed herein are certain structural requirements forperforming the disclosed functions, and it is understood that there area variety of structures that can perform the same function that arerelated to the disclosed structures, and that these structures willtypically achieve the same result.

B. Dendrimeric Compounds

Dendrimers can be ideal building blocks for biomedical applications,because of their precise architecture, high loading capacity, tunablesolubility, immunogenicity, and bioconjugation capability. [Gillies, E.R.; Frdchet, J. M. J. Drug Discov. Today 2005, 10, 35.; Lee, C. C.;MacKay, J. A.; Frdchet, J. M. J.; Szoka, F. C. Nat. Biotechnol. 2005,23, 1517.]The combination of the unique properties of dendrimers withmembrane-permeable guanidino groups can lead to a moreefficient-synthesis of membrane-permeable carrier molecules possessinghigh efficiency, for example, for bulk production.

The compounds of the invention are desirably based upon a compact, highbranching multiplicity dendrimer, for example, the classic Newkome-typedendrimer. [Newkome, G. R.; Behera, R. K.; Moorefield, C. N.; Baker, G.R. J. Org. Chem. 1991, 56, 7162.] Newkome type dendrimers are typically1→3 C-branched polyamide macromolecules, built from “Behera's Amine”monomer or its derivatives, and can be attached to a variety of startingcores, surfaces, and polymers.

It is also understood that the compounds of the invention can betailored to enhance accumulation in specific sublocations of cells, suchas the nucleus, the cytosol, or the mitochondria. Tailoring can be theselection of chemical moieties or groups having an affinity for atargeted subcellular region of a cell, for example an organelle, and thefunctionalization of the compounds with the selected chemical moietiesor groups. Such tailoring of the compound structure can be accomplishedusing organic synthetic methodology know to those of skill in the art.

In one aspect, the invention relates to compounds comprising thestructure:

and at least one guanidinium residue, wherein m is zero or a positiveinteger. In certain aspects, m can be 0, 1, 2, 3, 4, 5, or 6 and eachresidue can be substituted or unsubstituted. In a further aspect, m is1.

In one aspect, the invention relates to compounds comprising thestructure:

wherein n and o are, independently, zero or a positive integer; whereinR¹ and R² are, independently, hydrogen, oxygen, alkyl, acyl, thioacyl,or carbonyl; wherein R³ is hydrogen, alkyloxycarbonyl, or alkyl; R⁴ ishydrogen, or alkyloxycarbonyl; wherein R⁵ and R⁶ are, independently,hydrogen, or alkyl; and wherein R⁷ is hydrogen or alkyloxycarbonyl.

In a further aspect, the compounds can comprise the structure:

wherein n is an integer from 1 to 9; wherein R¹ and R² are,independently, hydrogen, oxygen, nitrogen, alkyl, acyl, thioacyl,carbonyl, or amine; wherein R³ is hydrogen or alkyl; and wherein R⁴ ishydrogen, or alkyloxycarbonyl, alkyl, or acyl. In certain aspects, n canbe 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9. In a further aspect, n is 1 or 5. Ina further aspect, R⁴ can be hydrogen or alkyloxycarbonyl. In a furtheraspect, R⁷ is Boc, for example, t-Boc.

In one aspect, the compound comprises the structure:

wherein n is an integer from 1 to 9; wherein R¹ and R² are,independently, hydrogen, amino, hydroxyl, alkyl, alkoxyl, acyl,carbonyl, or thioacyl; wherein R³ is hydrogen or alkyl; and wherein R⁴is hydrogen, or alkyloxycarbonyl.

C. Methods of Making Dendrimeric Compounds

The disclosed methods typically employ a divergent method to prepare aG-1 dendrimer scaffold with nine end functionalities. Although theNewkome type dendrimer is well known, one of the drawbacks for a broaderapplication of conventional methods is the elaborate synthesis of themonomer. In contrast, the “Behera's amine” gives the most compact, lowmolecular weight polyamide dendrimer possible; achieving the necessarynine end functionalities in just one generation of dendritic growth. Asset forth below and in the Experimental section, following synthesis ofthe monomer through improved hydrogenation and work-up procedures, theG-1 dendritic nona-acid scaffold can be prepared in high yields (seeFIGS. 6A and 6B).

In order to introduce the guanidinium groups to the dendrimer exterioras shown in FIG. 20, the nine carboxylic acid groups were firstconverted into nine protected amine groups, by reaction with, forexample, N-Boc ethylendiamine and N-Boc-1,6-diaminehexane through amidecoupling reactions. After removal of the protecting groups, the ninefree amines can be reacted with a guandinylating reagent [Feichtinger,K.; Sings, H. L.; Baker, T. J.; Matthews, K.; Goodman, M. J. Org. Chem.1998, 63, 8432.] to give a guanidinylated dendritic scaffold in highyield.

For uptake evaluation and imaging function, a fluorophore can beconjugated to the focal point of the molecular transporter. Theattachment of a fluorescein isothiocyanate (FITC) moiety to theguanidinylated scaffold can be achieved with a reduction of the nitrogroup at the focal point to an amino group via hydrogenation at roomtemperature in quantitative yields, followed by direct reaction withFITC to form the Boc-protected FITC-labeled guanidino-dendrimer. Afterdeprotection of the Boc-protected guanidine groups, FITC-labeleddendritic molecules can be obtained and further purified by dialysis orHPLC.

In one aspect, the invention relates to methods of preparing compoundshaving the structure:

wherein n is an integer from 1 to 9, wherein R³ is hydrogen or alkyl,wherein R⁴ and R⁷ are, independently, hydrogen, alkyloxycarbonyl, alkyl,or acyl; wherein R⁷ is hydrogen, alkyl, or acyl; wherein Y comprises anitro group, an amine group, an amide group, azide group, or analkyloxycarbonyl protected amine group or a derivative thereof, themethod comprising the steps of providing a first compound comprising thestructure:

wherein X comprises OH, halogen, or OC(O)-alkyl; coupling the firstcompound with at least about three molar equivalents of a secondcompound comprising the structure:

wherein G¹ is an ester-protecting group; removing the ester-protectinggroup; reacting the product of step (c) with at least about three molarequivalents of a third compound comprising the structure:

wherein G² is an amine-protecting group; removing the amine-protectinggroup; and functionalizing the product of step (e) with at least threemolar equivalents of a guanidine-providing agent.

In a further aspect, the guanidine-providing agent comprises at leastone of N,N′-diBoc-N″-triflylguanidine, N,N′-diCbz-N″-triflylguanidine,N,N′-dialloc-N″-triflylguanidine, N,N′-ditroc-N″-triflylguanidine,1,3-diboc-2-(2-hydroxyethyl)guanidine,N,N′-diBoc-1H-pyrazole-1-carboxamidine,N,N′-diCbz-1H-pyrazole-1-carboxamidine, 1H-pyrazole-1-carboxamidinehydrochloride, 1,3-diboc-2-(2-hydroxyethyl)guanidine,2-(2-aminoethyl)-1,3-diboc-guandine, or1,3-diboc-2-(carboxymethyl)guanidine

In a further aspect, the method further comprises the step oftransforming Y into an amine to provide a compound comprising thestructure:

In a further aspect, the method further comprises the step of removingR⁷. The removing step can be, for example, treatment with one or morereagents known to those of skill in the art for removing protectinggroups.

In one aspect, the providing step comprises synthesis of the startingmaterials. Each starting material can be obtained commercially and/orprepared by those of skill in the art from commercially availablecompounds. For example, the nitroester shown below can be prepared usingmethodology from Newkone, G. R.; Behera, R. K.; Moorefield, C. N.;Baker, G. R.; J. Org. Chem. 1991, 56, 7162:

In a further aspect, the ester-protecting group comprises methyl, ethyl,or t-butyl.

In a further aspect, the amine-protecting group comprises abutyloxycarbonyl group, a trifluoroacyl group, a9-fluorenylmethyloxycarbonyl group, an alloc group, or a carbobenzyloxygroup.

In a further aspect, the method further comprises the step of acylatingthe amine with a compound comprising the structure:

wherein o and p are, independently, zero or a positive integer. In a yetfurther aspect, the method further comprises the step of reacting theproduct of the acylating step with a payload compound comprising atleast one amine group and at least one of a luminescent group, abiologically active group, or a pharmaceutically active group.

In a further aspect, the method further comprises the step of acylatingthe amine with a fourth compound comprising the structure:

wherein o and p are, independently, zero or a positive integer, andwherein G³ is an thiol-protecting group.

In a further aspect, the thiol protecting group comprises the structure:

andwherein the fourth compound comprises the structure:

In a further aspect, the thiol-protecting group comprises the structure:

In a further aspect, the method further comprises the step of removingthe thiol-protecting group, thereby providing a deprotected thiol. In ayet further aspect, the method further comprises the step of attachingthe deprotected thiol to a thiol-functionalized payload. In a stillfurther aspect, the thiol-functionalized payload comprises at least oneof a luminescent group, a biologically-active group, or apharmaceutically-active group.

D. Compositions

In one aspect, the invention relates to compositions comprising one ormore compounds of the invention or one or more products of the methodsof the invention.

1. Intracellular Delivery Compositions

In one aspect, the invention relates to intracellular deliverycompositions comprising the general structure P-L-B-F, wherein P ispayload moiety; wherein L is a linking moiety comprising the structure:

wherein o and p are, independently, zero or a positive integer; whereinB is a branching moiety comprising the structure:

andwherein F is a functional moiety comprising at least one guanidiniumresidue. In a further aspect, p is an integer from 0 to 6, for example,0, 1, 2, 3, 4, 5, or 6. In a further aspect, the composition comprisesat least six guanidinium residues, at least seven guanidinium residues,at least eight guanidinium residues, or at least nine guanidiniumresidues.

In one aspect, L-B-F comprises the structure:

wherein n is an integer from 1 to 9; wherein R³ is hydrogen or alkyl;wherein R⁴ is hydrogen, alkyl or acyl; and wherein R⁷ is hydrogen, alkylor acyl.

In a further aspect, P-L-B-F comprises the structure:

wherein n is an integer from 1 to 9; wherein R³ is hydrogen or alkyl;wherein R⁴ is hydrogen, alkyl or acyl; wherein R⁷ is hydrogen, alkyl oracyl; and wherein R⁸ comprises the structure:

a. Payloads

Typically, the compounds of the invention can be functionalized to carrya payload. In various aspects, a payload compound can be attached orassociated with a compound of the invention by covalent bonding, byionic bonding, by coordination bonding, or by hydrogen bonding. Infurther aspects, a payload compound can be associated with a compound ofthe invention by hydrophilic interactions or hydrophobic interactions.In certain aspects, a payload compound is part of a compound of theinvention, while in certain further aspects, payload compound is aseparate compound from of a compound of the invention.

In one aspect, the payload moiety bears a thiol moiety. In a furtheraspect, the payload moiety is a luminescent group. For example, theluminescent group can comprise the structure:

In certain aspects, the luminescent group is selected from a dansylgroup, a coumarin group, an FITC group, a DOTA group, a catechol group,or a DPTA group. DOTA, catechol, and/or DPTA groups can be used forcomplexing, for example, lanthanides. Catechol can be used forcomplexing, for example, quantum dots, lanthanides, metals (such as ironor copper (e.g., radioactive Cu)), ironoxides, metal oxides, and/orplatinum (e.g., cis-platinum).

In a further aspect, the payload moiety is a biologically-active group.For example, the biologically-active group can be selected from one ormore of an oligonucleotide, a plasmid DNA, a protein, an immunoglobulin,an antisense oligoDNA, a peptide nucleic acid (PNA), or a peptide. Forexample, in various aspects, the biologically-active group can compriseone or more of β-galactosidase, horseradish peroxidase, RNase,anti-apoptotic proteins Bcl-X(L)/PEA-15, catalase, green fluorescenceprotein, heat shock protein 70, human glutamate dehydrogenase,ovalbumin, neuroptotectant Bcl-xL, E2 protein, phosphorothioateantisense oligonucleotides, anti-tetanus F(ab′)₂, G protein,p16^(INK4a), caspase-3, p14^(INK4a), p27^(kip1), Bak BH3 domain peptide,cGPK-Iα inhibitory peptide, IKKβ C-terminal peptide, PKA inhibitorypeptide, MEK 1 N-terminal peptide, luciferin, RhoA, APO-BEC-1, Crerecombinase, H-Ras, Filmin-1, p16, HPC-1/syntaxin, Cdk2, E2f-1/p73/p53,influenza virus, antibodies, single chain antibodies, si-RNA, RNAderivatives, peptide 46, peptide 15, peptides that influence theimmunresponse, mitochondrial DNA, bacteria, birdflu virus, and/orbacteria.

In a further aspect, the payload moiety is a pharmaceutically-activegroup. For example, the pharmaceutically-active group is selected from asmall molecular weight drug, a silica nanoparticle, a metalnanoparticle, a protein, a peptide, a linear polymer backbone, ahydrogel, a collapsed nanoparticle, a dendrimers, or a hyperbranchedpolymeric structure. For example, in various aspects, thepharmaceutically-active group can comprise one or more ofsuperparamagnetic iron oxide particles, doxorubicin, methotrexate,liposome, multiple sclerosis agents, cis-platinum, paclitaxel, hormones,antioxidants, antimicrobials, antibacterial agents, antidepressants,sedatives, antihypertensive drugs, antibodies, a carbohydrate-baseddrug, cardioprotective εPKC agonist peptide, Fab fragments of theanti-melanoma antibody NRML-05, pan-carcinoma antibody NRLU-10, anti-CEAimmunotoxin, liposome drugs, bromonidine, fusogenic, dendritic cellvaccines, VHL tumor suppressor peptide, HER-2, Pro-apotoxic Smacpeptide, viralcapsids, and/or bacteria.

A doxorubicin biocongujate, for example, can be synthesized as shown inFIG. 58.

In a still further aspect, the payload is an antibody, an intrabody,DNA, RNA, siRNA, among other biologically significant conjugates. Forexample, an antibody can be attached to the core of a discloseddendrimer, through disclosed methods. Such compositions can be used totreat viral related disorders, such as, for example, HIV or influenza,among others. A specific example of an antibody suitable for use withthe disclosed dendimers is an IgG antibody.

A disclosed dendrimer can also be attached to a protein is associatedwith a number of disorders, including cancer. For example, a disclosedprotein-dendrimer can conjugate can be used to treat a cancer. Anexample is a p53 (tumor suppressor protein) dendrimer conjugate whichcan be capable of restoration of a mutant p53 transcriptional activity,to trigger apoptosis and stop tumor progression through the cytoplasm. Afurther example is a dendrimer-Huntingtin (protein responsible ofHuntington's disease) conjugate which can aid in the inhibition ofaberrant protein aggregation in a cellular model of Huntington'sdisease, by targeting huntingtin to the nucleus, through the action ofthe dendritic molecular transporter.

Further examples of conjugates that can be used in combination with thedisclosed dendritic transporters include M and N intrabodies for RSV,RV6-26 Fab Rotavirus, Tat (HIV-1-transcription activator) for theinhibition of viral replication by sequestering Tat in the cytoplasm.

b. Intracellular Delivery

In one aspect, the invention relates to methods of intracellulardelivery comprising administering an effective amount of one or morecompounds of the invention or one or more compositions of the inventionto a subject. In one aspect, The subject is a mammal, for example, ahuman. In a further aspect, the subject is a cell. The delivery can be,for example, oral, transmucosal, rectal, or subcutaneous administrationor, for example, intravenous, intrathecal, intramuscular, intranasal,intraperitonel, or intraocular injection.

2. Pharmaceutical Compositions

A pharmaceutical composition comprising a therapeutically effectiveamount of one or more compounds of the invention or one or morecompositions of the invention and a pharmaceutically acceptable carrierfor administration in a mammal, for example, a human. The compositionscan be, for example, granules, powders, tablets, or capsules.

a. Dosage

The specific therapeutically effective dose level for any particularpatient will depend upon a variety of factors including the compound orcomposition being administered; the disorder being treated and theseverity of the disorder; the specific composition employed; the age,body weight, general health, sex and diet of the patient; the time ofadministration; the route of administration; the rate of excretion ofthe specific compound employed; the duration of the treatment; drugsused in combination or coincidental with the specific compound employedand like factors well known in the medical arts. If desired, theeffective daily dose can be divided into multiple doses for purposes ofadministration. Consequently, single dose compositions can contain suchamounts or submultiples thereof to make up the daily dose. The dosagecan be adjusted by the individual physician in the event of anycontraindications. Dosage can vary, and can be administered in one ormore dose administrations daily, for one or several days.

b. Carriers

A “pharmaceutically acceptable carrier” refers to sterile aqueous ornonaqueous solutions, dispersions, suspensions or emulsions, as well assterile powders for reconstitution into sterile injectable solutions ordispersions just prior to use. Examples of suitable aqueous andnonaqueous carriers, diluents, solvents or vehicles include water,ethanol, polyols (such as glycerol, propylene glycol, polyethyleneglycol and the like), carboxymethylcellulose and suitable mixturesthereof, vegetable oils (such as olive oil) and injectable organicesters such as ethyl oleate. Proper fluidity may be maintained, forexample, by the use of coating materials such as lecithin, by themaintenance of the required particle size in the case of dispersions andby the use of surfactants. These compositions may also contain adjuvantssuch as preservatives, wetting agents, emulsifying agents, anddispersing agents. Prevention of the action of microorganisms may beensured by the inclusion of various antibacterial and antifungal agentssuch as paraben, chlorobutanol, phenol, sorbic acid, and the like. Itcan also be desirable to include isotonic agents such as sugars, sodiumchloride, and the like. Prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of agents,such as aluminum monostearate and gelatin, which delay absorption.Injectable depot forms are made by forming microencapsule matrices ofthe drug in biodegradable polymers such as polylactide-polyglycolide,poly(orthoesters) and poly(anhydrides). Depending upon the ratio of drugto polymer and the nature of the particular polymer employed, the rateof drug release can be controlled. Depot injectable formulations arealso prepared by entrapping the drug in liposomes or microemulsionswhich are compatible with body tissues. The injectable formulations maybe sterilized, for example, by filtration through a bacterial-retainingfilter or by incorporating sterilizing agents in the form of sterilesolid compositions which can be dissolved or dispersed in sterile wateror other sterile injectable media just prior to use. Suitable inertcarriers can include sugars such as lactose. Desirably, at least 95% byweight of the particles of the active ingredient have an effectiveparticle size in the range of 0.01 to 10 micrometers.

E. Synthesis and Characterization of “Bow-Tie” Dendritic MolecularTransporters by Orthogonal and Click Approach

Disclosed is the synthesis and characterization of “Bow-Tie” dendriticarchitectures with orthogonally reactive groups, defined composition andfunctionality, which can be used as multi-drug carries for specificintracellular delivery. Huisgen cycloadditions or so called “click”reactions have been shown to be extremely versatile tools for advancedmacromolecular design. However, little attempt has been made to utilizethis approach to prepare multifunctional dendritic structures. In thedisclosed approach, two orthogonal protected dendritic structures arecombined by utilizing the “click” reaction. This strategy allows thecontrolled deprotection of the trifluoro protecting group to selectivelyattach the dithiopyridylpropionic acid the periphery of themacromolecule. In a further step, the BOC groups of the second dendriticscaffolds are deprotected to be guanydilated to the ethyl- or hexyllinker of the system. The bow-tie structure is the first of its kindthat consists of a molecular transporter part and drug delivery entityon the other. The chemistry applied for the construction ishigh-yielding and, thus, gives the bow-tie delivery structure in themost straightforward approach. In this fashion, nine drug molecules, forexample peptides, genes and oligonucleotides can be transported acrosscellular membranes.

Synthetic Pathway of Acid-labile Azide-linker-Dendron:

Synthetic Pathway for Base-labile Alkyne-linker-Dendron:

Bifunctional Bow-Tie Synthesis by Click Reaction:

Further Functionalization for Synthesis of Cell-Permeable Multi-DrugCarrier Conjugates:

In one aspect, the invention relates to compounds comprising thestructure:

wherein each m is independently zero or a positive integer, and whereinL is a linking moiety comprising optionally substituted alkyl,optionally substituted alkoxyl, optionally substituted heteroalkyl, oroptionally substituted heteroaryl.

In a further aspect, L comprises a structure:

wherein each n is independently selected from 0-8. That is, L cancomprise the reaction product of a “click” reaction.

In a further aspect, the compound can comprise a structure

wherein n is an integer from 1 to 9; wherein R³ is hydrogen or alkyl;wherein R⁴ is hydrogen, or alkyloxycarbonyl, alkyl, or acyl; and whereinR⁷ is hydrogen or alkyloxycarbonyl.

In a yet further aspect, the compound can comprise the structure:

wherein n is an integer from 1 to 9; wherein R³ is hydrogen or alkyl;wherein R⁴ is hydrogen, or alkyloxycarbonyl, alkyl, or acyl; and whereinR is hydrogen or alkyloxycarbonyl.

In a still further aspect, the compound can comprise the structure:

wherein n is an integer from 1 to 9; wherein R³ is hydrogen or alkyl;wherein R⁴ is hydrogen, or alkyloxycarbonyl, alkyl, or acyl; and whereinR⁷ is hydrogen or alkyloxycarbonyl.

In an even further aspect, the compound can comprise the structure:

wherein each n is independently an integer from 0 to 9; wherein R³ ishydrogen or alkyl; wherein R⁴ is hydrogen, or alkyloxycarbonyl, alkyl,or acyl; and wherein R⁷ is hydrogen or alkyloxycarbonyl.

It is demonstrated that the disclosed transporter (e.g., FD-2, hexyllinker) shows selectivity towards the mitochondria of a cell. (see FIG.33) The FD-1 shows selectivity towards the cell nucleus (see FIG. 32). Acommon obstacle in macromolecular drug delivery is the cellular uptakeinto cell compartments that do not release the drug delivery vector intothe cytosol or mitochondria in which the drug becomes effective. Mostother delivery pathways into the cell end up in the lysosome and do notget released (endocytosis). The therapeutic efficacy of drug moleculestypically depends on its ability to reach desired target tissues, cellsand intracellular organelles.

The mitochondria play a key role in apoptosis (cancer therapy), familialamyotrophic lateral sclerosis (ALS, Lou Gehrig's disease), Leberhereditary optic neuropathy (LHON), lactic acidosis, strokelike syndrome(MELAS) Huntington's disease, and Alzheimer's disease, Kearns-SayreSyndrome (KSS), myoclonic epilepsy, ragged-red fibers (MERRF), clusterof metabolic diseases (SyndromeX), progressive external ophthalmophlegia(PEO) and antioxidants. By targeting the mitochondria, the disclosedcompounds, compositions, and methods can play a role in therapy orprevention of disease processes relating to mitochondria function.

When the disclosed transporter is attached to the disclosednanoparticle, it can enter the cell and also can achieve localization inthe entire cell, including the mitochondria. The nanoparticle allowsdelivering a high drug load and, thus, can facilitate delivery of smalland other molecules, such as peptides, nucleotides and such. Thestructures can be further modified with amines to allow complexationwith plasmic DNA and covalent attachment s though covalent approaches.(See FIGS. 34-39).

A nanoparticle with a number of molecular transporter moietiesconjugated to the periphery crosses the plasmic membrane and localizesin the cytosol and, particularly, in the mitochondria of the cells.Techniques are disclosed that allow the attachment of the moleculartransporter the scaffolds that increase the drug load significantly.Attachment to the “bow-tie” structure and/or the attachment tonanoparticles from intramolecular chain collapse techniques alsoincrease the drug load significantly.

The dendritic transporter allows the conjugation of nine bioactiveconjugates and the drug load is increased nine fold by attaching adendric molecule to the focal point of the dendritic moleculartransporter (bow-tie). A well-defined macromolecule is designed, that is“clicked” together in a Huisgen type reaction. The deprotection of thebasic/acidic protecting groups allows the modification to a deliverysystem with a short ethyl linker or hexyl linker before guanidylation tomaintain uptake into specific subcellular locations. The disulfidelinker is only one of the examples of a linker chemistry attached to thedrug part of the bow-tie structure. All other linkers presented can beapplied as well.

The drug load can be increased nine-fold by attaching a dendric moleculeto the focal point of the dendritic molecular transporter (bow-tie). Awell-defined macromolecule is designed, that is “clicked” together in aHuisgen type reaction. The deprotection of the basic/acidic protectinggroups allows the modification to a delivery system with a short ethyllinker or hexyl linker before guanidylation to maintain uptake intospecific subcellular locations.

Here, the drug load can be increased to a theoretical amount of 100-300positions to conjugate small molecule drugs, peptides, oligonucleotidesand more. The functionalization of the particle with a varied amount ofamines allows together with the attachment of transporter allows thedevelopment of a gene delivery system. A “drug” can also be conjugatedthough a disulfide bond in a covalent conjugation approach. For example,proteins can be delivered. (See FIGS. 47, 48, 50, and 51).

F. Crosslinked Degradable Polymeric Nanoparticles

Traditional polyester nanoparticle delivery systems are typicallyself-assembled from linear polyesters chains driven by the polarity ofthe solvent, emulsion composition and addition techniques. Theseprocedures predetermine the drug loading during nanoparticle formationand limit post-modification chemistries in organic and aqueoussolutions. Furthermore, the result of this self-assembly process ismirrored in the morphology and degradation properties of the releasesystems. It has been recognized that the degradation behavior of thenanoparticles and release profile of the entrapped drug molecules arefactors to establish predictable pharmacokinetic profiles in effectivemultidrug cancer therapies. So far, release kinetics are challenged by arapid release of the drug molecules in the first 24-48 h followed by aslower release, referred to as a “burst-effect.” These release profilestypically prevent the establishment of reliable dosages and contributeto developing multidrug resistance, often times the result ofnon-optimized drug concentrations at tumor sites.

In contrast, actively targeted drug delivery carriers can entrap highconcentrations of hydrophobic therapeutics and maintain a linear releaseprofile, which can be tuned to the demands of the tumor type as a resultof the adjustable supramolecular architecture accomplished through anintermolecular cross-linking technique. The disclosed methods ofpreparing polyester particles utilize a controlled cross-linkingmechanism of linear polyester precursors that contain pendant functionalgroups as one of the cross-linking units with a difunctionalized linkerthat acts as the second cross-linking partner. To achieve control over aseries of different nanoparticle size dimensions, the amount of thedifunctionalized linker is added in a series of varying equivalencies tothe pendant functionalities of the linear polyester precursor.Nanoparticles can be produced, depending on the linker amount present inthe reaction, with unique sizes and standard deviations of only 10%.These “nano-networks,” depending on their nanoparticle size andcross-linking density, influence their crystallinity, but the particlesare amorphous at the temperature of use (37° C.). To determine if theamorphous properties of poly(valerolactoneepoxyvalerolactone),poly(vl-evl) particles have a positive effect on the degradationbehavior, a series of degradation studies in buffer at pH 7.4 at 37° C.were performed, investigating particles from a completed series oflinear precursors and increasing amounts of difunctionalizedcross-linkers with controlled nanoscopic dimensions (FIG. 1).

Degradation of the particles was monitored by the change of the absolutemolecular weight, as determined through static light scattering (SLS).Linear degradation profiles were observed for all particlesinvestigated, with the highest loss of molecular weight for the 725 nmnanoparticle with 17.5% of its total molecular mass remaining after 10days. Smaller particles with a slightly higher degree of crystallinityof 20.6% were degraded to 26% of the original molecular weight. Theobserved linear degradation kinetics are a parameter that determines thequality of the developed particles towards applications as controlledrelease systems.

The capacity to encapsulate small molecule drugs, such as paclitaxel(taxol), can also be evaluated. Traditional polyester particles,produced with salting-out or nanoprecipitation methods, typically do notexceed a drug loading over 5% that is facilitated during nanoparticleformation. However, the disclosed nanoparticles consist of crosslinkedsupramolecular structures that are readily soluble in organic solventswithout affecting the 3-D architecture. This property provides theopportunity to load the particles after formation by dissolving theparticles in dimethyl sulfoxide (DMSO) together with cancertherapeutics, such as paclitaxel (taxol), and precipitating into water.

Determination of drug loading capacity was performed with particles of53 nm in diameter from linear precursors,poly(-valerolactone-epoxyvalerolactone-allylvalerolactone-oxepanedione),poly(vl-evl-avl-opd), containing 11% epoxide and crosslinked with 2equivalents of diamines per epoxide (FIG. 3). In preparation for in vivoexperiments, the encapsulation method was designed to also increase thehomogenity of the particle dispersion in water for a practicaladministration of the drug loaded particles by injection. Anemulsification process with vitamin E TPGS (D-a-tocopherol polyethyleneglycol 1000 succinate) was used, which achieves a homogenous dispersionof the loaded or un-loaded particles in water or buffer. The resultingparticles are analyzed by UV-Vis with a NanoDrop Spectrophotometer at254 nm, and along with a calibration curve, the drug loading withpaclitaxel was found to be 15.7% for an aimed 20% drug load and 11.3%for a 15% drug load, respectively. With this process, it is not onlypossible to load therapeutic drug molecules to a higher degree intoprepared nanoparticles, but it is also possible to solubilizehydrophobic cancer therapeutics in aqueous solutions.

Side effects known to be caused by adjuvant agents, such as Cremophor EL(50:50 ethanol-polyoxyethylated castor oil) to solubilize hydrophobicdrug molecules for intravenous injections, can be avoided. To ensurethat no cellular toxicity is caused by the vitamin E TPGS formulatedparticles prior to drug loading, the cell viability was assessed byutilizing a MTT assay (FIG. 2). The cellular toxicity was determined byincubating HeLa cells with varying concentrations of particles intriplicate ranging from 5 mg/ml to 0.001 mg/ml. Following 24 h ofincubation with particles, cell viability was assessed. As seen in FIG.2, the nanoparticles did not cause significant cytotoxicity against theHeLa cell line. The experimental TC50 value for the formulated particleswas found to be 1.0 mg/ml. Moreover, emulsification had an effect on thedegradation profile and was found to correlate with the in vitro releasestudies. Over the period of 16 days, the particles experienced a lowcontrolled degradation, as seen by the linear degradation profile,finishing with 70% of its original molecular weight remaining (FIG. 3).Without wishing to be bound by theory, it is believed that the slowerdegradation rate can be attributed to the well-defined structure of thenanoparticle and the vitamin E TPGS that remains at the surface tostabilize the particles. Consequently, this gradual constant degradationprofile of the particles is a desirable feature, as it translates intothe controlled and sustained release of therapeutics.

The paclitaxel release kinetics from vitamin E TPGS formulatednanoparticles were assessed by monitoring the cumulative release oftaxol at 37° C. in DPBS at pH 7.4. At particular time intervals, thesamples were centrifuged, and the supernatant was taken for analysis ofpaclitaxel concentration by NanoDrop spectrophotometry (254 nm). FIG. 4depicts the cumulative release of paclitaxel from the particles. Theprofile shows a collective release of 4.4% and 7.4% taxol in the first 2and 6 h respectively, followed by a slow and sustained release over 60days, which again confirmed the efficient encapsulation of paclitaxelwithin the cross-linked nanoparticles. Without wishing to be bound bytheory, it is believed that the initial instant release of paclitaxel inthe first several hours is due to the dissolution or diffusion of thedrug that was absorbed onto the nanoparticle surface, while the linearslow continuous release is attributed to the diffusion of the drugencapsulated in the nanoparticle during degradation. In contrast,traditional poly(lactic-co-glycolic acid) (PLGA) nanoparticlesexperience an erratic nonlinear drug release, that includes a“burst-effect” in which about 40% of taxol is released in the first day,followed by a fast release of about 10-30% in the next 2-5 days and thenfinally a slow release till no paxlitaxel (taxol) remains. In isnoteworthy that the release kinetics can be adjusted to faster or slowerrelease, governed by the density of cross-linking and the particle size.

In preparation for in vivo studies, in which the particle sizes can playan role in the interaction with the tumor vasculature, the influence offormulation and encapsulation of small molecule drugs to the diameter ofthe nanoparticles was evaluated. It was found that based on the 3-Dcrosslinked network structure, the size dimension slightly changes from53 nm to 57 nm and indicates the conformity of the 3-D network structureupon encapsulation, as seen by transmission electron microscopy (TEM),(FIG. 5), with 2-8 times more drug incorporated compared to traditionalpolyester nanoparticle systems. Drug release profiles for conventionalpolyester based nanoparticle systems, compared to the disclosednanoparticle systems, are shown in FIG. 8.

Thus, the disclosed nanoparticle synthesis pathways allow for theintroduction of functional groups, such as alkyne, allyl or ketofunctionalities, that are not affected by the cross-linking reactionsand nanoparticle formation. In particular, thiol-ene “click” reactionsallow for the conjugation of peptides with integrated cysteines added tothe sequence near the N-terminus. Such mild reaction conditions do notrequire the addition of radical starters and use slightly elevatedtemperatures of 37° C. To synthesize the drug delivery systems, thelinear poly(vl-evl-avl-opd) precursor was prepared, which wascross-linked with 2 equivalents of diamines per epoxide to form ananoparticle of 53 nm in size. The remaining allyl groups were thenfunctionalized with peptides to target radiated and nonradiated tumorvasculature, such as the reported peptides with recognition unitsHVGGSSV and cRGD, respectively (FIG. 6). The bioconjugates were analyzedvia NMR, DLS and SLS and were then loaded with paclitaxel and formulatedwith vitamin E. Using UV-Vis, the loading capacity was found to be 11%,aiming for a 15% drug load.

It can also be demonstrated that the linear release kinetics are in factadjustable. With decreasing cross-linking density, the drug molecule isreleased at a higher rate. By decreasing the cross-linking density(˜50%), from 7% to 2%, the release rate increased by around 50%. Thesedata point indicate that a 15% cross-linking can decrease the release byanother 50% with 140 days for 100% release and would afford a very slowrelease rate. To afford a faster release of the drug molecule than the2% cross-liking, a longer cross-linker (MW 2003) can be used to preparea particle with a wider network architecture for an even faster releaseprofile. With a 7% cross-linking density, 40% of the drug is released in6 days. It is understood that with the decrease of the cross-linkingdensity to 2%, the release can be increased to 3 days (40%). This isrepresented schematically in FIG. 9.

1. Polymers

It is understood that the disclosed polymers can be used in connectionwith the disclosed nanoparticles and disclosed methods. Unless stated tothe contrary, the disclosed structures can be used in connection withthe disclosed methods, the disclosed polymers, and the disclosednanoparticles.

a. Epoxide-Functionalized Polymers

In one aspect, the invention relates to a polymer comprising at leastone monomer residue having an optionally substituted structurerepresented by a formula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl; wherein m is aninteger from 0 to 6; wherein n is an integer from 0 to 2; and whereinthe monomer residue comprises less than about 10% by weight of themonomer residue of halogen selected from chlorine, bromine, and iodine.In further aspects, the monomer residue can comprise less than about 8%,less than about 5%, less than about 4%, less than about 3%, less thanabout 2%, or less than about 1% of halogen selected from chlorine,bromine, and iodine, by weight of the monomer residue.

In a further aspect, an epoxide-functionalized polymer can furthercomprise at least one monomer residue selected from apropargyl-functionalized monomer residue having an optionallysubstituted structure represented by a formula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, wherein m¹ is aninteger from 0 to 6, and wherein n¹ is an integer from 0 to 2; a monomerresidue having an optionally substituted structure represented by aformula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, and wherein n²is an integer from 0 to 2; and a keto-functionalized monomer residuehaving an optionally substituted structure represented by a formula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, and wherein n³is an integer from 0 to 2.

In one aspect, Z is O. That is, the polymer residue can be a polyesterresidue. In a further aspect, the polymer is a polyester. In a furtheraspect, the polymer is a co-polyester.

In a further aspect, the Z is NR, wherein R is H or C1 to C6 alkyl. Inone aspect, the polymer residue can be a polyamide residue. In a furtheraspect, the polymer is a polyamide. In a further aspect, the polymer isa co-polyamide. The alkyl can be optionally further substituted. R canbe C1 to C6, C2 to C6, C1 to C5, C2 to C5, C1 to C4, C2 to C4, C1, C2,C3, C4, C5, or C6 alkyl.

In one aspect, the polymer comprises at least one monomer residue havingan optionally substituted structure represented by a formula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl; wherein Y is O,S, or NR, wherein R is H or C1 to C6 alkyl; wherein R^(L) is selectedfrom optionally substituted alkyl and optionally substituted alkoxylene;wherein m is an integer from 0 to 6; and wherein n is an integer from 0to 2.

In a further aspect, the polymer further comprises at least one monomerresidue selected from a propargyl-functionalized monomer residue havingan optionally substituted structure represented by a formula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, wherein m¹ is aninteger from 0 to 6, and wherein n¹ is an integer from 0 to 2; a monomerresidue having an optionally substituted structure represented by aformula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, and wherein n²is an integer from 0 to 2; and a keto-functionalized monomer residuehaving an optionally substituted structure represented by a formula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, and wherein n³is an integer from 0 to 2.

In a further aspect, the polymer comprises an optionally substitutedstructure represented by a formula:

wherein m and m′ are independently integers from 0 to 6; wherein n andn′ are independently integers from 0 to 2; and wherein Z and Z′ areindependently O or NR, wherein R is H or C1 to C6 alkyl; wherein Y andY′ are independently O, S, or NR, wherein R is H or C1 to C6 alkyl; andwherein R^(L) is selected from optionally substituted alkyl andoptionally substituted alkoxylene.

In various aspects, m can be an integer from 0 to 6, from 1 to 6, from 0to 5, from 1 to 5, from 0 to 4, from 1 to 4, from 0 to 3, from 1 to 3,from 0 to 2, from 1 to 2, 0, 1, 2, 3, 4, 5, or 6. In various aspects, m′can be an integer from 0 to 6, from 1 to 6, from 0 to 5, from 1 to 5,from 0 to 4, from 1 to 4, from 0 to 3, from 1 to 3, from 0 to 2, from 1to 2, 0, 1, 2, 3, 4, 5, or 6. In various aspects, m¹ can be an integerfrom 0 to 6, from 1 to 6, from 0 to 5, from 1 to 5, from 0 to 4, from 1to 4, from 0 to 3, from 1 to 3, from 0 to 2, from 1 to 2, 0, 1, 2, 3, 4,5, or 6.

In various aspects, n can be an integer from 0 to 2, from 1 to 2, from 0to 1, 0, 1, or 2. In various aspects, n′ can be an integer from 0 to 2,from 1 to 2, from 0 to 1, 0, 1, or 2. In various aspects, n¹ can be aninteger from 0 to 2, from 1 to 2, from 0 to 1, 0, 1, or 2. In variousaspects, n² can be an integer from 0 to 2, from 1 to 2, from 0 to 1, 0,1, or 2. In various aspects, n³ can be an integer from 0 to 2, from 1 to2, from 0 to 1, 0, 1, or 2.

R^(L) can be selected from optionally substituted alkyl and optionallysubstituted alkoxylene. Suitable alkyls include divalent organicradicals selected from ethyl, propyl, butyl, pentyl, hexyl, heptyl,octyl, decyl, dodecyl, hexadecyl, and higher alkyl. Suitable alkoxyleneinclude divalent organic radicals selected from groups having astructure represented by a formula:

Further suitable alkoxylene include divalent organic radicals selectedfrom groups having a structure represented by a formula:

Further suitable alkoxylene include a divalent organic radical having astructure represented by a formula:

which can be derived from 2,2-(ethylenedioxy)bis(ethylamine).

The polymers and copolymers typically have a number average molecularweight (Mn) of from about 3500-4800 Daltons with a narrow polydispersityof from about 1.17 to about 1.27. It is understood that the molecularweight can be higher or lower and that one of skill in the art canreadily manipulate reaction conditions to achieve a different desiredmolecular weight.

b. Multifunctional Polymers

In one aspect, a polymer can be a multifunctional polymer. That is, thepolymer comprises monomer residues selected from two or more of anepoxide-functionalized monomer residue having an optionally substitutedstructure represented by a formula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl; wherein m is aninteger from 0 to 6; wherein n is an integer from 0 to 2; and apropargyl-functionalized monomer residue having an optionallysubstituted structure represented by a formula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, wherein m¹ is aninteger from 0 to 6, and wherein n¹ is an integer from 0 to 2; and aketo-functionalized monomer residue having an optionally substitutedstructure represented by a formula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, and wherein n³is an integer from 0 to 2.

In one aspect, the epoxide-functionalized monomer residue is present andcomprises less than about 10% by weight of the monomer residue ofhalogen selected from chlorine, bromine, and iodine. In further aspects,the monomer residue can comprise less than about 8%, less than about 5%,less than about 4%, less than about 3%, less than about 2%, or less thanabout 1% of halogen selected from chlorine, bromine, and iodine, byweight of the monomer residue.

In a further aspect, the polymer further comprises at least one monomerresidue having an optionally substituted structure represented by aformula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, and wherein n²is an integer from 0 to 2.

In one aspect, a polymer comprises at least one monomer residue havingan optionally substituted structure represented by a formula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, wherein Y is O,S, or NR, wherein R is H or C1 to C6 alkyl, wherein R^(L) is selectedfrom optionally substituted alkyl and optionally substituted alkoxylene,wherein m is an integer from 0 to 6, and wherein n is an integer from 0to 2; and one or more of:a propargyl-functionalized monomer residue having an optionallysubstituted structure represented by a formula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, wherein m¹ is aninteger from 0 to 6, and wherein n¹ is an integer from 0 to 2; anda keto-functionalized monomer residue having an optionally substitutedstructure represented by a formula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, and wherein n³is an integer from 0 to 2. In a further aspect, the polymer furthercomprises at least one monomer residue having an optionally substitutedstructure represented by a formula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, and wherein n²is an integer from 0 to 2. In one aspect, the at least one monomerresidue has an optionally substituted structure represented by aformula:

wherein m and m′ are independently integers from 0 to 6; wherein n andn′ are independently integers from 0 to 2; and wherein Z and Z′ areindependently O or NR, wherein R is H or C1 to C6 alkyl; wherein Y andY′ are independently O, S, or NR, wherein R is H or C1 to C6 alkyl; andwherein R^(L) is selected from optionally substituted alkyl andoptionally substituted alkoxylene.

In one aspect, at least one monomer residue has an optionallysubstituted structure represented by a formula:

wherein m and m¹ are independently integers from 0 to 6; wherein n andn¹′ are independently integers from 0 to 2; and wherein Z and Z′ areindependently O or NR, wherein R is H or C1 to C6 alkyl; wherein Y is O,S, or NR, wherein R is H or C1 to C6 alkyl; and wherein R^(L) isselected from optionally substituted alkyl and optionally substitutedalkoxylene.

In various aspects, m can be an integer from 0 to 6, from 1 to 6, from 0to 5, from 1 to 5, from 0 to 4, from 1 to 4, from 0 to 3, from 1 to 3,from 0 to 2, from 1 to 2, 0, 1, 2, 3, 4, 5, or 6. In various aspects, m′can be an integer from 0 to 6, from 1 to 6, from 0 to 5, from 1 to 5,from 0 to 4, from 1 to 4, from 0 to 3, from 1 to 3, from 0 to 2, from 1to 2, 0, 1, 2, 3, 4, 5, or 6. In various aspects, m¹ can be an integerfrom 0 to 6, from 1 to 6, from 0 to 5, from 1 to 5, from 0 to 4, from 1to 4, from 0 to 3, from 1 to 3, from 0 to 2, from 1 to 2, 0, 1, 2, 3, 4,5, or 6. In various aspects, m¹ can be an integer from 0 to 6, from 1 to6, from 0 to 5, from 1 to 5, from 0 to 4, from 1 to 4, from 0 to 3, from1 to 3, from 0 to 2, from 1 to 2, 0, 1, 2, 3, 4, 5, or 6.

In various aspects, n can be an integer from 0 to 2, from 1 to 2, from 0to 1, 0, 1, or 2. In various aspects, n′ can be an integer from 0 to 2,from 1 to 2, from 0 to 1, 0, 1, or 2. In various aspects, n¹ can be aninteger from 0 to 2, from 1 to 2, from 0 to 1, 0, 1, or 2. In variousaspects, n^(1′) can be an integer from 0 to 2, from 1 to 2, from 0 to 1,0, 1, or 2. In various aspects, n² can be an integer from 0 to 2, from 1to 2, from 0 to 1, 0, 1, or 2. In various aspects, n³ can be an integerfrom 0 to 2, from 1 to 2, from 0 to 1, 0, 1, or 2.

R^(L) can be selected from optionally substituted alkyl and optionallysubstituted alkoxylene. Suitable alkyls include divalent organicradicals selected from ethyl, propyl, butyl, pentyl, hexyl, heptyl,octyl, decyl, dodecyl, hexadecyl, and higher alkyl. Suitable alkoxyleneinclude divalent organic radicals selected from groups having astructure represented by a formula:

Further suitable alkoxylene include divalent organic radicals selectedfrom groups having a structure represented by a formula:

Further suitable alkoxylene include a divalent organic radical having astructure represented by a formula:

which can be derived from 2,2-(ethylenedioxy)bis(ethylamine) or2,2-(ethylenedioxy)bis(ethylazide).

In one aspect, a polymer can comprise at least one monomer residuehaving an optionally substituted structure represented by a formula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, wherein R^(L) isselected from optionally substituted alkyl and optionally substitutedalkoxylene, wherein m¹ is an integer from 0 to 6, and wherein n is aninteger from 0 to 2; and one or more of:an epoxide-functionalized monomer residue having an optionallysubstituted structure represented by a formula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl; wherein m is aninteger from 0 to 6; wherein n is an integer from 0 to 2; anda keto-functionalized monomer residue having an optionally substitutedstructure represented by a formula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, and wherein n³is an integer from 0 to 2. In a further aspect, theepoxide-functionalized monomer residue is present and comprises lessthan about 10% by weight of the monomer residue of halogen selected fromchlorine, bromine, and iodine.

In one aspect, the polymer further comprises at least one monomerresidue having an optionally substituted structure represented by aformula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, and wherein n²is an integer from 0 to 2.

In a further aspect, at least one monomer residue has an optionallysubstituted structure represented by a formula:

wherein m¹ and m¹ are independently integers from 0 to 6; wherein n¹ andn¹ are independently integers from 0 to 2; and wherein Z and Z′ areindependently O or NR, wherein R is H or C1 to C6 alkyl; and whereinR^(L) is selected from optionally substituted alkyl and optionallysubstituted alkoxylene.

In one aspect, at least one monomer residue has an optionallysubstituted structure represented by a formula:

wherein m¹ and m′ are independently integers from 0 to 6; wherein n¹ andn′ are independently integers from 0 to 2; and wherein Z and Z′ areindependently O or NR, wherein R is H or C1 to C6 alkyl; and wherein Y′is O, S, or NR, wherein R is H or C1 to C6 alkyl; wherein R^(L) isselected from optionally substituted alkyl and optionally substitutedalkoxylene.

2. Nanoparticles

It is understood that the disclosed nanoparticles can be used inconnection with the disclosed polymers and disclosed methods. Unlessstated to the contrary, the disclosed structures can be used inconnection with the disclosed methods, the disclosed polymers, and thedisclosed nanoparticles.

In one aspect, the invention relates to a degradable polymericnanoparticle comprising at least one monomer residue having anoptionally substituted structure represented by a formula:

wherein m and m′ are independently integers from 0 to 6; wherein n andn′ are independently integers from 0 to 2; and wherein Z and Z′ areindependently O or NR, wherein R is H or C1 to C6 alkyl; wherein Y andY′ are independently O, S, or NR, wherein R is H or C1 to C6 alkyl; andwherein R^(L) is selected from optionally substituted alkyl andoptionally substituted alkoxylene, wherein the nanoparticle has aparticle size of from about 5 nm to about 850 nm.

In one aspect, the nanoparticle further comprises at least one monomerresidue selected from a propargyl-functionalized monomer residue havingan optionally substituted structure represented by a formula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, wherein m¹ is aninteger from 0 to 6, and wherein n¹ is an integer from 0 to 2; aketo-functionalized monomer residue having an optionally substitutedstructure represented by a formula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, and wherein n³is an integer from 0 to 2; and a monomer residue having an optionallysubstituted structure represented by a formula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, and wherein n²is an integer from 0 to 2. In a further aspect, Z and Z′ are 0.

In one aspect, the nanoparticle further comprises at least oneepoxide-functionalized monomer residue having an optionally substitutedstructure represented by a formula:

In a further aspect, the nanoparticle further comprises at least onefunctionalized monomer residue having an optionally substitutedstructure represented by a formula:

wherein X is OH, SH, NH₂, or NHR, wherein R is H or C1 to C6 alkyl; andwherein R¹ is an optionally substituted organic radical comprising 1 to24 carbon atoms.

In a further aspect, R¹ is further substituted with at least onebiologically active agent, at least one pharmaceutically active agent,and/or at least one imaging moiety.

In one aspect, the nanoparticle further comprises at least onenucleophile-functionalized monomer residue having an optionallysubstituted structure represented by a formula:

In a further aspect, the nanoparticle further comprises at least onefunctionalized monomer residue having an optionally substitutedstructure represented by a formula:

wherein R¹ is an optionally substituted organic radical comprising 1 to24 carbon atoms.

In a further aspect, R¹ is further substituted with at least onebiologically active agent, at least one pharmaceutically active agent,and/or at least one imaging moiety.

In one aspect, the invention relates to a degradable polymericnanoparticle comprising at least one monomer residue having anoptionally substituted structure represented by a formula:

wherein m¹ and m^(1′) are independently integers from 0 to 6; wherein n¹and n^(1′) are independently integers from 0 to 2; and wherein Z and Z′are independently O or NR, wherein R is H or C1 to C6 alkyl; and whereinR^(L) is selected from optionally substituted alkyl and optionallysubstituted alkoxylene, wherein the nanoparticle has a particle size offrom about 5 nm to about 850 nm.

In a further aspect, the nanoparticle further comprises at least onemonomer residue selected from: an epoxide-functionalized monomer residuehaving an optionally substituted structure represented by a formula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl; wherein m is aninteger from 0 to 6; wherein n is an integer from 0 to 2; aketo-functionalized monomer residue having an optionally substitutedstructure represented by a formula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, and wherein n³is an integer from 0 to 2; and a monomer residue having an optionallysubstituted structure represented by a formula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, and wherein n²is an integer from 0 to 2. In a further aspect, Z and Z′ are O.

In a further aspect, the nanoparticle further comprises at least onepropargyl-functionalized monomer residue having an optionallysubstituted structure represented by a formula:

In a further aspect, the nanoparticle further comprises at least onefunctionalized monomer residue having an optionally substitutedstructure represented by a formula:

wherein R¹ is an optionally substituted organic radical comprising 1 to24 carbon atoms. In a further aspect, R¹ is further substituted with atleast one biologically active agent, at least one pharmaceuticallyactive agent, and/or at least one imaging moiety.

In a further aspect, the nanoparticle further comprises at least oneazide-functionalized monomer residue having an optionally substitutedstructure represented by a formula:

In a further aspect, the nanoparticle further comprises at least onefunctionalized monomer residue having an optionally substitutedstructure represented by a formula:

wherein R¹ is an optionally substituted organic radical comprising 1 to24 carbon atoms. In a further aspect, R¹ is further substituted with atleast one biologically active agent, at least one pharmaceuticallyactive agent, and/or at least one imaging moiety.

In one aspect, the invention relates to crosslinked degradablenanoparticles having a polyester backbone and one or more crosslinkshaving a structure selected from:

wherein Y is O, S, or N—R, wherein R is C1-C4 alkyl;

wherein L is a divalent alkyl chain or alkyloxyalkyl chain.

In a further aspect, the one or more crosslinks are produced by anucleophilic epoxide ring opening reaction. In a further aspect, the oneor more crosslinks are produced by a reductive amination reaction. In afurther aspect, the one or more crosslinks are produced by an azidealkyne cycloaddition.

In a further aspect, the nanoparticle further comprises one or morebiologically active agents or pharmaceutically active agents.

In a further aspect, the nanoparticle is produced by crosslinking apolymer comprising at least one monomer residue having an optionallysubstituted structure represented by a formula:

wherein m is an integer from 0 to 6, and wherein n is an integer from 0to 2; or at least one propargyl-functionalized monomer residue having anoptionally substituted structure represented by a formula:

wherein m¹ is an integer from 0 to 6, and wherein n¹ is an integer from0 to 2; or at least one monomer residue having an optionally substitutedstructure represented by a formula:

wherein n² is an integer from 0 to 2; or at least oneketo-functionalized monomer residue having an optionally substitutedstructure represented by a formula:

wherein n³ is an integer from 0 to 2.

In one aspect, the invention relates to compositions comprising adegradable polyester nanoparticle and, encapsulated therein, abiologically active agent, a pharmaceutically active agent, or animaging agent. In a further aspect, the biologically active agent isencapsulated within the nanoparticle. In a further aspect, thepharmaceutically active agent is encapsulated within the nanoparticle.In a further aspect, the imaging agent is encapsulated within thenanoparticle.

In a further aspect, the degradable polyester nanoparticle comprises acrosslinked degradable nanoparticle having a polyester backbone and oneor more crosslinks having a structure selected from:

wherein Y is O, S, or N—R, wherein R is C1-C4 alkyl;

wherein L is a divalent alkyl chain or alkyloxyalkyl chain.

G. Preparation Methods

It is understood that the disclosed methods can be used in connectionwith the disclosed polymers and disclosed nanoparticles. Unless statedto the contrary, the disclosed structures can be used in connection withthe disclosed methods, the disclosed polymers, and the disclosednanoparticles.

1. Methods of Making Polymer

To address the deficiencies of conventional nanoparticle compositionsand methods, the availability of novel functional polyesters that alloworthogonal modification approaches was addressed. Additionally,controlled chain cross-linking strategies for obtaining distinctnanoparticles in a variety of nanoscopic dimensions are disclosed. Incontrast to investigating emulsification-solvent techniques [Hans, M.L.; Lowman, A. M. Curr. Opin. Solid State Mater. Sci. 2002, 6, 319-327.]or emulsion diffusion methods [Kallinteri, P.; Higgins, S.; Hutcheon, G.A.; St. Pourcain, C. B.; Garnett, M. C. Biomacromolecules 2005, 6,1885-1894.] that need surfactants or salts, the disclosed methods andcompositions involve controlled cross-linking techniques.

A clean and non-toxic cross-linking entity can be provided from epoxidegroups that react with dinucleophiles (e.g., diamines) to form alkane—OH groups. While this crosslinking unit has been employed to formacrylate based microparticles [Burke, S. K.; Slatopolsky, E. A.;Goldberg, D. I., Nephrol. Dial. Transplant. 1997, 12, (8), 1640-1644.],it has been never investigated in the formation of degradablenanoparticles due to the lack of suitable linear precursors.

The epoxide entity for the formation of discrete cross-linkednanoparticles can be integrated by polymerization of a low molecularweight linear copolymer, Ab, with pendant allyl groups. See FIG. 10.Pendant allyl groups represent valuable intermediates to many functionalgroups and can be incorporated into the polymer backbone bycopolymerizing α-allyl-δ-valerolactone, (b), and commercially availableδ-valerolactone, (A), via ring-opening polymerization (ROP). [Parrish,B.; Quansah, J. K.; Emrick, T. J. Polym. Sci. Part A: Polym. Chem. 2002,40, 1983-1990.] Upon copolymerization, the pendant allyl groups can beoxidized by a Baeyer-Villiger oxidation with meta-chloroperbenzoic acid(m-CPBA) to convert the double bonds to epoxide rings, which then becamea coupling group in the preparation of the nanoparticles. [(a)Mecerreyes, D.; Miller, R. D.; Hedrick, J. L.; Detrembleur, C.; Jerome,R. J. Polym. Sci. Part A: Polym. Chem. 2000, 38, 870-875. (b) Latere, J.P.; Lecomte, P.; Dubois, P.; Jérôme, R. Macromolecules 2002, 35,7857-7859.] To introduce additional functional groups into thenanoparticle, additional monomers can be synthesized, for exampleα-propargyl-δ-valerolactone, (C), and 2-oxepane-1,5-dione, (D). Thesemonomers can then be individually copolymerized with (B) and8-valerolactone, (A), in a similar manner as Ab, to give rise to linearpolyesters with additional propargyl or keto functionalitiesrespectively. To increase the number of functionalities that alloworthogonal modification approaches, (C) and (D) were copolymerizedtogether with (b) and δ-valerolactone (A), as summarized in FIG. 10. Thecopolymers were typically obtained in molecular weight ranges of3500-4800 Da with narrow polydispersities of 1.17-1.27.

In one aspect, the invention relates to a method of preparing a polymercomprising the step of copolymerizing a mixture of two or more of analkene-functionalized monomer providing a residue having an optionallysubstituted structure represented by a formula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl; wherein m is aninteger from 0 to 6; wherein n is an integer from 0 to 2; apropargyl-functionalized monomer providing a residue having anoptionally substituted structure represented by a formula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, wherein m¹ is aninteger from 0 to 6, and wherein n¹ is an integer from 0 to 2; and aketo-functionalized monomer providing a residue having an optionallysubstituted structure represented by a formula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, and wherein n³is an integer from 0 to 2.

In a further aspect, the mixture further comprises at least one monomerproviding a residue having an optionally substituted structurerepresented by a formula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, and wherein n²is an integer from 0 to 2.

In one aspect, the alkene-functionalized monomer is present and themethod further comprises the step of oxidizing the resultant polymer toprovide an epoxide-functionalized monomer residue having an optionallysubstituted structure represented by a formula:

In a further aspect, the alkene-functionalized monomer is present andhas an optionally substituted structure represented by a formula:

In a further aspect, the propargyl-functionalized monomer is present andhas an optionally substituted structure represented by a formula:

In a further aspect, the keto-functionalized monomer is present and hasan optionally substituted structure represented by a formula:

In a further aspect, the monomer providing a residue having anoptionally substituted structure represented by a formula:

has an optionally substituted structure represented by a formula:

In one aspect, the invention relates to a method of preparing anepoxide-functionalized polymer comprising the step of oxidizing apolymer having at least one monomer residue having an optionallysubstituted structure represented by a formula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, wherein m is aninteger from 0 to 6, and wherein n is an integer from 0 to 2.

In a further aspect, the polymer further comprises at least one monomerresidue selected from:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, wherein m¹ is aninteger from 0 to 6, and wherein n¹ is an integer from 0 to 2;

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, wherein n² is aninteger from 0 to 2; and

wherein n³ is an integer from 0 to 2.

In a further aspect, at least one monomer residue has an optionallysubstituted structure represented by a formula:

wherein m is an integer from 0 to 6, and wherein n is an integer from 0to 2. For example, in one aspect, m is 1, and n is 0, providing anoptionally substituted structure represented by a formula:

In a further aspect, the epoxide-functionalized polymer has anoptionally substituted structure represented by a formula:

2. Methods of Crosslinking

In one aspect, the invention relates to a method of crosslinking apolymer comprising the step of reacting a polymer comprising at leastone monomer residue selected from an epoxide-functionalized monomerresidue having an optionally substituted structure represented by aformula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl; wherein m is aninteger from 0 to 6; wherein n is an integer from 0 to 2; and apropargyl-functionalized monomer residue having an optionallysubstituted structure represented by a formula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, wherein m¹ is aninteger from 0 to 6, and wherein n¹ is an integer from 0 to 2; with across-linker having a structure represented by a formula X—R^(L)—X′,wherein X and X′ are independently N₃, OH, SH, NH₂, or NHR, wherein R isH or C1 to C6 alkyl, and wherein R^(L) is selected from optionallysubstituted alkyl and optionally substituted alkoxylene.

In one aspect, the linker groups can be bis-nucleophilic (e.g., diamine)compounds derived from alkylene oxides (e.g., diamino poly(ethyleneoxides)) and/or alkyls (e.g., 1,8-diaminooctane; Jeffamines) and theirderivatives.

In a further aspect, the linker groups can be thiols. For example, thedinucleophile can have a structure X—R^(L)—X′, wherein X and X′ are eachSH, wherein R is H or C1 to C6 alkyl, and wherein R^(L) is selected fromoptionally substituted alkyl, optionally substituted alkoxylene, andoptionally substituted esters.

Thiols suitable for crosslinking include mono- and di-thiol analogues ofcompounds derived from alkylene oxides (e.g., diamino poly(ethyleneoxides)) and/or alkyls (e.g., 1,8-diaminooctane; Jeffamines) and theirderivatives. Other suitable dithiols for cross-linking include:

An example crosslinking reaction, and example product thereof, is shownin FIG. 59.

In one aspect, the cross-linker reacts with two polymer strands. In afurther aspect, X—R^(L)—X′ reacts with two epoxide-functionalizedmonomer residues. In a further aspect, X—R^(L)—X′ reacts with twopropargyl-functionalized monomer residues. In a further aspect,X—R^(L)—X′ reacts with one epoxide-functionalized monomer residue andone propargyl-functionalized monomer residue. In a further aspect, X═X′.In a further aspect, X═X′═NH₂. In a further aspect, R^(L) comprises twoor more residues of ethylene oxide or trimethylene oxide. In a furtheraspect, X—R^(L)—X′ is 2,2-(ethylenedioxy)bis(ethylamine). In a furtheraspect, X═X′═N₃.

In one aspect, the polymer comprises at least one monomer residue havingan optionally substituted structure represented by a formula:

and wherein X═X′═NH₂. In one aspect, the polymer and the crosslinker arereacted in a ratio of about 1:1 (polymer:cross-linker). In a furtheraspect, the polymer and the crosslinker are reacted in a ratio ofabout >1:1 (polymer:cross-linker) to provide a polymer with excessepoxide-functionalization. In a further aspect, the polymer and thecrosslinker are reacted in a ratio of about <1:1 (polymer:cross-linker)to provide a polymer with excess amino-functionalization.

In one aspect, the polymer comprises at least one monomer residue havingan optionally substituted structure represented by a formula:

and wherein X═X′═N₃. In a further aspect, the polymer and thecrosslinker are reacted in a ratio of about 1:1 (polymer:cross-linker).In a further aspect, the polymer and the crosslinker are reacted in aratio of about >1:1 (polymer:cross-linker) to provide a polymer withexcess alkyne-functionalization. In a further aspect, the polymer andthe crosslinker are reacted in a ratio of about <1:1(polymer:cross-linker) to provide a polymer with excessazide-functionalization.

In a further aspect, the polymer further comprises a keto-functionalizedmonomer providing a residue having an optionally substituted structurerepresented by a formula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, and wherein n³is an integer from 0 to 2.

In a further aspect, the polymer further comprises at least one monomerproviding a residue having an optionally substituted structurerepresented by a formula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, and wherein n²is an integer from 0 to 2.

3. Methods of Functionalizing Polymers

In one aspect, the invention relates to a method of functionalizing apolymer comprising the step of reacting an epoxide-functionalizedmonomer residue having an optionally substituted structure representedby a formula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl; wherein m is aninteger from 0 to 6; wherein n is an integer from 0 to 2; with anucleophile having a structure represented by a formula X—R¹, wherein Xis OH, SH, NH₂, or NHR, wherein R is H or C1 to C6 alkyl; and wherein R¹is an optionally substituted organic radical comprising 1 to 24 carbonatoms.

Organic radicals suitable for use as R¹ include substituted orunsubstituted monovalent organic radicals selected from ethyl, propyl,butyl, pentyl, hexyl, heptyl, octyl, decyl, dodecyl, hexadecyl, andhigher alkyl. The alkyl can be linear or branched and can be cyclic oracyclic. In a further aspect, R¹ can comprise an optionally substitutedalkoxylene. Suitable alkoxylene include substituted or unsubstitutedmonovalent organic radicals selected from groups having a structurerepresented by a formula:

wherein R³ comprises C1 to C6 alkyl.

In a further aspect, R¹ is further substituted with at least onebiologically active agent, at least one pharmaceutically active agent,and/or at least one imaging moiety, thus providing a convenient methodfor functionalizing the polymer with one or more biologically activeagents, pharmaceutically active agents, and/or imaging moieties via anucleophilic substitution reaction. That is, R¹ can comprise at leastone biologically active agent, at least one pharmaceutically activeagent, and/or at least one imaging moiety. In a further aspect, R¹ cancomprise a portion of the at least one biologically active agent, atleast one pharmaceutically active agent, and/or at least one imagingmoiety. In a further aspect, R¹ can be covalently bonded to at least onebiologically active agent, at least one pharmaceutically active agent,and/or at least one imaging moiety.

In one aspect, the invention relates to a method of functionalizing apolymer comprising the step of reacting a propargyl-functionalizedmonomer residue having an optionally substituted structure representedby a formula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, wherein m¹ is aninteger from 0 to 6, and wherein n¹ is an integer from 0 to 2; with anazide having a structure represented by a formula N₃—R¹, wherein R¹ isan optionally substituted organic radical comprising 1 to 24 carbonatoms. In a further aspect, R¹ is further substituted with at least onebiologically active agent, at least one pharmaceutically active agent,and/or at least one imaging moiety.

In one aspect, the invention relates to a method of functionalizing apolymer comprising the steps of reacting a keto-functionalized monomerproviding a residue having an optionally substituted structurerepresented by a formula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, and wherein n isan integer from 0 to 2; with an amine having a structure represented bya formula H₂N—R¹, wherein R¹ is an optionally substituted organicradical comprising 1 to 24 carbon atoms; and reducing the resultingimine. In a further aspect, the reacting step and the reducing step areperformed simultaneously. In a further aspect, R¹ is further substitutedwith at least one biologically active agent, at least onepharmaceutically active agent, and/or at least one imaging moiety.

In one aspect, the invention relates to a method of functionalizing apolymer comprising the step of reacting a nucleophile-functionalizedmonomer residue having an optionally substituted structure representedby a formula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl; wherein m is aninteger from 0 to 6; wherein n is an integer from 0 to 2; wherein Y andY′ are independently O, S, or NR, wherein R is H or C1 to C6 alkyl; andwherein R^(L) is selected from optionally substituted alkyl andoptionally substituted alkoxylene; with an electrophile having astructure represented by a formula E-R¹, wherein E is an electrophilicmoiety; and wherein R¹ is an optionally substituted organic radicalcomprising 1 to 24 carbon atoms.

In a further aspect, Y′ is NH₂ or NHR. In a further aspect, whereinY═Y′. In a further aspect, the electrophilic moiety is selected fromalkyl halide, alkyl pseudohalide, and carboxyl derivative. In a furtheraspect, R¹ is further substituted with at least one biologically activeagent, at least one pharmaceutically active agent, and/or at least oneimaging moiety.

4. Methods of Making Nanoparticles

The formation of nanoparticles in controlled size dimensions can proceedfrom linear polymers containing pendant epoxide groups which crosslinkwith 2,2′-(ethylenedioxy)bis(ethylamine). To evaluate the particleformation under controlled conditions, reactions in which theequivalents of diamine cross-linker were linearly increased with respectto the reactive epoxide groups of the polymers were studied.

To achieve a high degree of cross-linking between the individualpolyester chains, the polymer solution with the pendant expoxideentities can be added in a dropwise fashion to a refluxing solution ofdifferent equivalents of dinucleophile (e.g., diamine) indichloromethane. In this strategy, the difunctional amine is in highexcess during the addition (13 mL/min) of the linear polymer solution(0.5 M) and thus provides optimum cross-linking reactions (Table 1;particle size reported in nm diameter by dynamic light scattering (DLS)in relation to varying amine ratios).

TABLE 1 Nanoparticle Size Dimensions (nm) Diameter (nm) Diameter (nm)Diameter (nm) Poly(vl-evl) Poly(vl-evl-opd) Poly(vl-evl-pvl) Amine/1Epoxide AB ABD ABC 1 30.71 ± 2.21 34.29 ± 3.22 21.40 ± 2.90 2 58.06 ±6.20 63.46 ± 7.68 41.70 ± 5.36 3  82.1 ± 5.73 118.3 ± 13.6 114.9 ± 8.9 4 115.6 ± 25.4 164.9 ± 65.7 148.3 ± 25.2 5 255.7 ± 60.3 292.7 ± 80.3186.1 ± 37.5 6 342.2 ± 52.2 341.0 ± 86.6 253.9 ± 41.4 8 425.1 ± 100 525.0 ± 100   472.1 ± 103.1 10 725.1 ± 94.3 800.0 ± 135   675.0 ± 126.1Diameter (nm) M_(w, RI) M_(w) Amine/1 Epoxide AB₁ nanoparticles^(a)(g/mol)^(b) PDI^(c) (kg/mol)^(d) 1 30.71 ± 2.21 3403 1.16 60.5 ± 3.5 258.06 ± 6.20 3445 1.16 81.5 ± 4.6 3 82.61 ± 5.73 3544 1.17 96.1 ± 4.9 4115.6 ± 12.5 3860 1.18 112 ± 6  5 255.7 ± 26.9 4005 1.18 187 ± 8  6342.2 ± 42.2 4267 1.21 222 ± 11 8 425.1 ± 44.6 4470 1.21 328 ± 15 10725.1 ± 94.3 4887 1.22 525 ± 28

The first trial was employed with polymer (AB) and implemented 1 to 10equivalents of amine functionalities to the pendant epoxidecross-linking entity. The resulting particles were characterized bytransmission electron microscopy (TEM) that provides the actual size,and by dynamic light scattering (DLS), to obtain the hydrodynamicdiameter as a representative measure of the particle under physiologicalconditions. Micrographs of representative nanoparticles are shown inFIG. 11. It is also contemplated that reaction stoichiometry can beselected to utilize in excess often (10) equivalents, thereby providingmicroparticles, materials for us in tissue engineering and biogels inbiomedical applications and devices.

As illustrated in FIG. 12, the particle size increase with a polynominaltrend as the equivalents of amine rises. For example, two equivalents ofamine yielded 58 nm particles, and five equivalents produced particleswith 255 nm dimensions (Table 1). Synthesized linear polymers containingadditional functionalities (ABC and ABD) were found to respond in thesame way to the controlled intermolecular chain crosslinking conditions,as with polymer (AB) from the original trial, and well-definednanoparticles were obtained (Table 1). As shown in FIG. 13,characterization of the particles with ¹H NMR confirmed the nanoparticleformation for each trial with an increase of signals at 3.5 and 2.89 ppmcorresponding to protons neighboring the secondary amine of thepolyethylene glycol (PEG)-linker after successful crosslinking event. Inparticular, a shift in resonance from 2.86 to 2.89 ppm was observed dueto the change of the primary amine to the secondary amine aftercross-linking. As a consequence, the continuous increase in aminecross-linker equivalents not only extends the particle size, but it alsointroduces additional amine functionalities connected to short PEGlinker that are available for further modification strategies.

In one aspect, the invention relates to a method of preparing adegradable nanoparticle comprising the step of adding a polymercomprising at least one monomer residue having a structure representedby a formula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, wherein m is aninteger from 0 to 6, and wherein n is an integer from 0 to 2; to asolution of from about 1 to about 10 molar equivalents of adinucleophile (nucleophilic moiety:epoxide functionality) having astructure X—R^(L)—X′, wherein X and X′ are independently OH, SH, NH₂, orNHR, wherein R is H or C1 to C6 alkyl, and wherein R^(L) is selectedfrom optionally substituted alkyl and optionally substituted alkoxylene.In a further aspect, the monomer residue comprises less than about 10%by weight of the monomer residue of halogen selected from chlorine,bromine, and iodine. In a further aspect, Z is O.

In one aspect, the solution comprises from about 1 molar equivalent of adinucleophile (nucleophilic moiety:epoxide functionality) and theresultant nanoparticle has a particle size of from about 5 nm to about55 nm. In a further aspect, the solution comprises from about 1 molarequivalent of a dinucleophile (nucleophilic moiety:epoxidefunctionality) and the resultant nanoparticle has a particle size offrom about 5 nm to about 55 nm. In a further aspect, the solutioncomprises from about 2 molar equivalents of a dinucleophile(nucleophilic moiety:epoxide functionality) and the resultantnanoparticle has a particle size of from about 30 nm to about 80 nm. Ina further aspect, the solution comprises from about 3 molar equivalentsof a dinucleophile (nucleophilic moiety:epoxide functionality) and theresultant nanoparticle has a particle size of from about 70 nm to about120 nm. In a further aspect, the solution comprises from about 4 molarequivalents of a dinucleophile (nucleophilic moiety:epoxidefunctionality) and the resultant nanoparticle has a particle size offrom about 110 nm to about 170 nm. In a further aspect, the solutioncomprises from about 5 molar equivalents of a dinucleophile(nucleophilic moiety:epoxide functionality) and the resultantnanoparticle has a particle size of from about 175 nm to about 300 nm.In a further aspect, the solution comprises from about 6 molarequivalents of a dinucleophile (nucleophilic moiety:epoxidefunctionality) and the resultant nanoparticle has a particle size offrom about 250 nm to about 350 nm. In a further aspect, the solutioncomprises from about 8 molar equivalents of a dinucleophile(nucleophilic moiety:epoxide functionality) and the resultantnanoparticle has a particle size of from about 400 nm to about 550 nm.In a further aspect, the solution comprises from about 10 molarequivalents of a dinucleophile (nucleophilic moiety:epoxidefunctionality) and the resultant nanoparticle has a particle size offrom about 650 nm to about 850 nm. It is also contemplated that reactionstoichiometry can be selected to utilize in excess of ten (10) molarequivalents, thereby providing higher particle sizes.

In one aspect, the invention relates to a method of preparing adegradable nanoparticle comprising the step of adding a polymercomprising at least one monomer residue having a structure representedby a formula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, wherein m¹ is aninteger from 0 to 6, and wherein n¹ is an integer from 0 to 2; to asolution of from about 1 to about 10 molar equivalents of a bis-azide(azide moiety:alkyne functionality) having a structure N₃—R^(L)—N₃,wherein R^(L) is selected from optionally substituted alkyl andoptionally substituted alkoxylene. In a further aspect, the monomerresidue comprises less than about 10% by weight of the monomer residueof halogen selected from chlorine, bromine, and iodine. In a furtheraspect, Z is O.

In one aspect, the invention relates to a method of controlling particlesize during the preparation of a degradable nanoparticle comprising thestep of adding an epoxide-functionalized polymer to a solution of adinucleophilic cross-linker, wherein the stoichiometry of thecross-linker (ratio of nucleophilic moiety:epoxide functionality) isselected to provide a desired particle size according to one or more ofthe graphs shown in FIG. 14-FIG. 19.

5. Methods of Functionalizing Nanoparticles

In one aspect, the invention relates to a method of functionalizing ananoparticle comprising the step of reacting a nanoparticle comprisingat least one epoxide-functionalized monomer residue having an optionallysubstituted structure represented by a formula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl; wherein m is aninteger from 0 to 6; wherein n is an integer from 0 to 2; with anucleophile having a structure represented by a formula X—R¹, wherein Xis OH, SH, NH₂, or NHR, wherein R is H or C1 to C6 alkyl; and wherein R¹is an optionally substituted organic radical comprising 1 to 24 carbonatoms. In a further aspect, R¹ is further substituted with at least onebiologically active agent, at least one pharmaceutically active agent,and/or at least one imaging moiety.

In one aspect, the invention relates to a method of functionalizing ananoparticle comprising the step of reacting a nanoparticle comprisingat least one propargyl-functionalized monomer residue having anoptionally substituted structure represented by a formula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, wherein m is aninteger from 0 to 6, and wherein n¹ is an integer from 0 to 2; with anazide having a structure represented by a formula N₃—R¹, wherein R¹ isan optionally substituted organic radical comprising 1 to 24 carbonatoms. In a further aspect, R¹ is further substituted with at least onebiologically active agent, at least one pharmaceutically active agent,and/or at least one imaging moiety.

In one aspect, the invention relates to a method of functionalizing ananoparticle comprising the steps of reacting a nanoparticle comprisingat least one keto-functionalized monomer providing a residue having anoptionally substituted structure represented by a formula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl, and wherein n³is an integer from 0 to 2; with an amine having a structure representedby a formula H₂N—R¹, wherein R¹ is an optionally substituted organicradical comprising 1 to 24 carbon atoms; and reducing the resultingimine. In a further aspect, the reacting step and the reducing step areperformed simultaneously. In a further aspect, R¹ is further substitutedwith at least one biologically active agent, at least onepharmaceutically active agent, and/or at least one imaging moiety.

In one aspect, the invention relates to a method of functionalizing ananoparticle comprising the step of reacting a nanoparticle comprisingat least one nucleophile-functionalized monomer residue having anoptionally substituted structure represented by a formula:

wherein Z is O or NR, wherein R is H or C1 to C6 alkyl; wherein m is aninteger from 0 to 6; wherein n is an integer from 0 to 2; wherein Y andY′ are independently O, S, or NR, wherein R is H or C1 to C6 alkyl; andwherein R^(L) is selected from optionally substituted alkyl andoptionally substituted alkoxylene; with an electrophile having astructure represented by a formula E-R¹, wherein E is an electrophilicmoiety; and wherein R¹ is an optionally substituted organic radicalcomprising 1 to 24 carbon atoms. In a further aspect, Y′ is NH₂ or NHR.In a further aspect, Y═Y′. In a further aspect, the electrophilic moietyis selected from alkyl halide, alkyl pseudohalide, and carboxylderivative. In a further aspect, R¹ is further substituted with at leastone biologically active agent, at least one pharmaceutically activeagent, and/or at least one imaging moiety.

6. Methods of Degrading Nanoparticles

In one aspect, the invention relates to a method of degrading adegradable nanoparticle comprising subjecting the nanoparticle toreaction conditions sufficient to hydrolyze an ester. In a furtheraspect, the conditions are biological conditions. In a further aspect,the conditions involve exposure to an esterase. In a further aspect, theconditions exist within an organism.

In one aspect, the invention relates to a method of degrading adegradable polymer comprising subjecting the polymer to reactionconditions sufficient to hydrolyze an ester. In certain aspects, thedegradable polymer is a disclosed polymer or a product of a disclosedmethod.

H. Functionalized Polymers and Nanoparticles

In one aspect, the disclosed nanoparticles can be functionalized with,for example, the disclosed dendrimeric compounds. That is, in oneaspect, the invention relates to a nanoparticle-dendrimer conjugate. Ina further aspect, the nanoparticle can be a disclosed organic quantumdots via intramolecular chain collapse. In a further aspect, thenanoparticle can be a disclosed degradable nanoparticle. In a furtheraspect, the dendrimer can be a disclosed intracellular deliverycomposition.

As disclosed herein, certain nanoparticles can bear electrophilic (e.g.,ketone) functionalities. Vinylsulfonyl functionality can be introducedto the disclosed nanoparticles. Thus, a vinylsulfonyl linker moiety wasprepared that can be attached in a reductive amination procedure to aketo groups of the particle. The synthesis of such a linker appears inScheme 1 in FIG. 60. It is understood that the alkyl chain can behomologated by selection of appropriate reagents.

The vinylsulfonyl moiety readily reacts with a nucleophile (e.g., athiol) to form a covalent bond, thereby further functionalizing ananoparticle. These linkers can be used to attach peptides that arelabeled with dye molecules at the focal point of the peptide or otheramines groups of the peptide. The thiol groups of cysteines can be usedto attach to the vinyl function of the vinyl sulfonyl linker. Also, thethiol group in the focal point of the disclosed dendritic moleculartransporters can be attached to the vinyl sulfonyl (or allyl) group,thus allowing a transporter to be attached to any post-modifiednanoparticle.

The same reaction can be used to attach peptides that are not labeledwith dye. In such cases, the particle can be labeled with dye or notlabeled.

Peptides (or other amines) can also be attached directly through theamine terminus of the peptide to the keto group through reductiveamination. See Scheme 2 in FIG. 61. Here, it is preferred that thepeptide contains only one amine group. Before the reductive amination isperformed, the particle can be labeled with a dye that adds to the aminefunctionality of the particle. After the reaction, residual dyes can bequenched so as to not interfere with the following reductive amination.

Similar systems can be constructed with particles from intramolecularcross-linking reactions. Replacing N-BED with an ethylenoxide equivalentenhances the solubility of the system.

Another approach that can enable formation of nanoparticle-dendrimerconjugate involves direct attachment of nucleophile-functionalizedmoieties (e.g., peptides or disclosed intracellular deliverycompositions) to an allylic function on disclosed degradablenanoparticles. As shown in Scheme 4, direct attachment of a thiol withan allyl functionalized polymer or nanoparticle can bypass use of thedisclosed SVEC linker.

In one aspect, an allylic function on disclosed degradable nanoparticlescan be provided via incomplete oxidation of epoxide functionalities, asshown in Scheme 5a.

Still other examples of linear precursors can be prepared according toScheme 5b.

An allyl functionality is thus available for functionalization andallows very mild conditions for the attachment of peptides and othermoieties that contain nucleophilic (e.g., thiol) groups. The allylgroups from Ab linear precursors can be partially preserved by partialoxidation to the epoxide that is needed for cross-linking to thenanoparticle to from AbB linear polymer. This chemistry is alsocompatible with the keto-group-containing ABD linear precursor to fromAbBD.

The nanoparticle formation does not take part in the cross-linkingreaction and is therefore available for further modification. The allylgroup is inert under the conditions used during the cross-linkingprocess. The crosslinking reaction is illustrated in Scheme 6 in FIG.63.

Again, the thiol group of the focal point of the dendritic moleculartransporter can be attached to the allyl group. One advantage of suchattachment is that it requires no other reagent. This can permit thetransporter to be attached to any already post-modified nanoparticlebecause of the mild reaction conditions.

In order to track the drug delivery system and study the uptake intotissues, an imaging moiety (e.g., a dye molecule such as rodamine orother dye) that has functionality to react with amines such as NHS-esteror isothiocyanates can be attached to the free amine groups that resultfrom the cross-linking reaction, as shown in Scheme 7 in FIG. 64. Theallyl groups or all other groups introduced are not affected.

The allyl groups can then be reacted with thiol groups of the focalpoint of the dendritic transporter, as illustrated in Scheme 8a in FIG.65, thereby providing multiply functionalized degradable nanoparticles.

The number of molecular transporter(s) bonded to the nanoparticle can beselected by varying the stoichiometry of the reagents added to the allylgroups. The same reaction can be performed with thiol groups attached topeptides. It was found that elevated temperatures such as 37° C. speedup the reaction but do not destroy the peptide.

In a further aspect, a nanoparticle can be attached to a discloseddendritic molecular transporter through an exemplary strategy shown inScheme 8b in FIG. 66.

The dendritic transporter shown in Scheme 8 can be furtherfunctionalized according to Scheme 8c in FIG. 67.

The nanoparticle of the intramolecular chain collapse reaction can bereacted with the commercially available N-Boc ethylenoxide amine. Theamine can be deprotected via acid cleavage with HCl or formic acid. Someof the free amines can be labeled with dye via NHS-ester reaction orthioisocyanide reaction. An SVEC moiety can then be connected through anNHS ester reaction. After the reaction the residual amine groups arebeing quenched. The thiol groups are attached to the vinylsulfone groupsof the SVEC. The thiol groups of the molecular transporter can also beattached in the same fashion as the peptides, as shown in Scheme 9a inFIG. 68.

Another example of attaching a peptide to a nanoparticle core is shownin Scheme 9b in FIG. 69.

Imaging moieties (e.g., dyes or DOTA moieties) that can function astherapeutic and tracking units can also be attached via a nucleophilicfunctionality, as shown in Scheme 10 in FIG. 70.

In a further aspect, a disclosed nanoparticle can be functionalized witha dye for imaging the eye in a subject. For example, such a method canbe accomplished conveniently by Scheme 12b in FIG. 73.

In a further aspect, analogous chemistry can be used to prepare a drugdelivery system comprising a drug molecule that is attached to a pHsensitive linker and includes a hydrazide linker and doxorubicin. Thesynthesis is illustrated in Schemes 13 and 14 and in Scheme 15 in FIG.74.

A novel c-RGD has been prepared and can be attached to the nanoparticlesand used for targeting of the disclosed delivery systems (See Scheme 16in FIG. 75).

The synthesis of the c-RGD that contains free amine and thiol unit forattachment to SVEC of the particle from the intra-molecular chaincollapse and the SVEC or the allyl group of the polyester particles isdetailed in Scheme 17 in FIG. 76.

The attachment of the molecular transporter to the maleiminde of theintra-molecular chain collapse particle has also been investigated tocreate a system that transports peptides to intracellular location andacross biological barriers. See Scheme 18.

Further modifications of the nanoparticle-dendrimer conjugate systemshave also been investigated. See Schemes 19-20. The disclosedmodifications, as well as analogous transformations, results in acollection of compounds available for use in intracellular transport.

I. Nanosponges

In one aspect, the invention relates to intravitreal drug-deliverynanoparticles (“nanosponges”), which are three-dimensional nano-networksformed from degradable materials, in particular, formed by crosslinkingdegradable linear polyesters. In various aspects, nanosponges can referto compositions comprising one or more disclosed compounds of theinvention or one or more products of the disclosed methods. Inparticular, nanosponges can refer to disclosed compounds or productsencapsulating one or more pharmaceutically active agent or biologicallyactive agent, for example, agents disclosed herein.

In a further aspect, a nanosponge is an ocular delivery platform(degradable polyester nanoparticle pharmaceutical or biologically activeagent complex, which can be also referred to as a nanoparticle complex,and can comprise one or more degradable crosslinked polyesternanoparticles and one or more biologically active agents, one or morepharmaceutically active agents, and/or one or more imaging agents, asdisclosed herein. In a particular aspect, a nanosponge is an oculardelivery platform for treatment and/or prevention of eye diseases (e.g.,glaucoma) and cancer (e.g., intraocular melanoma).

Nanosponges can offer significant advantages over conventional drugdelivery systems. For example, nanosponges can be prepared usingpractical synthetic methods in suitable nanoscopic dimensions. In oneaspect, nanosponges can be prepared for treatment of eye disease (e.g.,400 nm and 700 nm) or for treatment of cancer (e.g., 50 nm and,optionally, modified with targeting unit that only targets cancer site).

In one aspect, nanosponges can encapsulate hydrophobic, potent drugs aswell as solubilize them in high concentrations. This leads to a largerpool of drugs available for drug discovery efforts. It is observed thatthere is no accumulation of nanosponges in other organs. As disclosedherein, nanosponges can be tailored to facilitate treatment of cancertype and disease stage. For example, drug release can be tailored (e.g.,fast, medium, slow), which can be important for fast and slow growingcancer types (e.g., beast, prostate, lung, and brain). Nanosponges canbe prepared for release of the encapsulated drug at a constant rate,which can be important for the development of clinical protocols.

In one aspect, the disclosed nanosponges can be used in connection withtreatment of eye diseases such as glaucoma (4th major cause ofblindness): Inter Ocular Pressure (IOP) can be controlled over a periodof two months with ONE treatment, so far limited or no treatmentpossible.

J. Methods of Administration

The disclosed compositions are useful for the deposition ofpharmaceutical agents encapsulated within the degradable polyesternanoparticle. Thus, disclosed herein are methods of administering apharmaceutical or biologically active agent to a cell comprisingcontacting the cell with a degradable polyesternanoparticle-pharmaceutical or biologically active agent complex(nanoparticle complex) thereby administering the pharmaceuticalbiologically active agent to the cell. It is contemplated herein thatthe nanoparticles can release the pharmaceutical agents over time as theparticle degrades resulting in the time release of the agent.

It is understood the nanoparticle-pharmaceutical agent complex can beadministered to any cell type desired. For example, the cell can be aneuron (e.g., a photoreceptor neuron), ganglion cell, cone cell, rodcell, epithelial cell, muscle cell, adipose cell, hepatic cell,erythrocyte, leukocyte, mast cell, fibroblast (e.g., a cornealfibroblast). Such cells can be part of a larger tissue such as neuronal,fibrous, blood, gangloid, dermal, muscular, amacrine, bipolar,horizontal, connective, epithelial, and vitreal fluid. It is furtherunderstood that the cells to which the nanoparticle-pharmaceutical agentcomplex is applied can be located in a region of an organ such as theeye. Examples of such regions include by are not limited to a region ofthe eye selected from the group consisting of sclera, cornea, retina,vitrius fluid, rods, cones, iris, zonular fibers, aqueous humour,choroid, ciliary muscle, optic disc, dura mater, optic nerve, fovea, andmacula. Because the nanoparticle-pharmaceutical agent complex can bedelivered to living tissue, organs, or cells, it is further contemplatedherein that said complexes have particular uses for administration of apharmaceutical agent to a subject.

Due to the size of the nanoparticle complexes disclosed herein, it isunderstood that the degradable polyester nanoparticles can be used todeliver a pharmaceutical agent directly to the interior of a cell. Thuscontemplated herein are methods of administration, wherein thenanoparticle compex is administered to anorganelle of a cell such as forexample mitochondria, the nucleus, the golgi apparatus, endoplasmicreticulum, ribosomes, lysosomes, or centrioles. Thus, for example,disclosed herein are methods of administering a pharmaceutical orbiologically active agent to the nucleus comprising contacting a cellwith a degradable polyester nanoparticle-pharmaceutical or biologicallyactive agent complex. It is understood that the complex can be taken upby the cell or can pass through a molecular channel such that thepharmaceutical or biologically active agent is internalized into thecell. It is further understood that the nanoparticle complex can furtherpass through organelle membranes to enter mitochondria or the nucleus ofthe cell.

It is understood that there are circumstances where one of skill in theart would want to monitor the deposition of the pharmaceutical orbiologically active agent following the administration of the degradablepolyester nanoparticle pharmaceutical or biologically active complexes.Therefore, it is contemplated herein that the nanoparticle complexes canfurther comprise a mechanism for detection. Detection can occur the useof imaging agents such as labels and dyes, but can also occur throughthe measure of physical characteristics such as measuring interocularpressure (IOP) or visualization such as electron microscopy. Where a dyeor label is used, the means of detection can employ any method known inthe art including but not limited to microscopy such asimmunofluorescence, radioimmunoassay, ELISAs, ELISpot, and flowcytometry. As used herein, a label can include radiolabels, pigmentdyes, a fluorescent dye, a member of a binding pair, such asbiotin/streptavidin, a metal (e.g., gold), or an epitope tag that canspecifically interact with a molecule that can be detected, such as byproducing a colored substrate or fluorescence. Substances suitable fordetectably labeling proteins include fluorescent dyes (also known hereinas fluorochromes and fluorophores) and enzymes that react withcolorometric substrates (e.g., horseradish peroxidase). The use offluorescent dyes is generally preferred in the practice of the inventionas they can be detected at very low amounts. Furthermore, in the casewhere multiple antigens are reacted with a single array, each antigencan be labeled with a distinct fluorescent compound for simultaneousdetection. Labeled spots on the array are detected using a fluorimeter,the presence of a signal indicating an antigen bound to a specificantibody.

Fluorophores are compounds or molecules that luminesce. Typicallyfluorophores absorb electromagnetic energy at one wavelength and emitelectromagnetic energy at a second wavelength. Representativefluorophores include, but are not limited to, 1,5 IAEDANS; 1,8-ANS;4-Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein;5-Carboxyfluorescein (5-FAM); 5-Carboxynapthofluorescein;5-Carboxytetramethylrhodamine (5-TAMRA); 5-Hydroxy Tryptamine (5-HAT);5-ROX (carboxy-X-rhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE;7-Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD); 7-Hydroxy-4-Imethylcoumarin; 9-Amino-6-chloro-2-methoxyacridine (ACMA); ABQ; AcidFuchsin; Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin;Acriflavin Feulgen SITSA; Aequorin (Photoprotein); AFPs—AutoFluorescentProtein—(Quantum Biotechnologies) see sgGFP, sgBFP; Alexa Fluor 350™;Alexa Fluor 430™; Alexa Fluor 488™; Alexa Fluor 532™; Alexa Fluor 546™;Alexa Fluor 568™; Alexa Fluor 594™; Alexa Fluor 633™; Alexa Fluor 647™;Alexa Fluor 660™; Alexa Fluor 680™; Alizarin Complexon; Alizarin Red;Allophycocyanin (APC); AMC, AMCA-S; Aminomethylcoumarin (AMCA); AMCA-X;Aminoactinomycin D; Aminocoumarin; Anilin Blue; Anthrocyl stearate;APC-Cy7; APTRA-BTC; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R;Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine; ATTO-TAG™ CBQCA;ATTO-TAG™ FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9(Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); BerberineSulphate; Beta Lactamase; BFP blue shifted GFP (Y66H); Blue FluorescentProtein; BFP/GFP FRET; Bimane; Bisbenzemide; Bisbenzimide (Hoechst);bis-BTC; Blancophor FFG; Blancophor SV; BOBO™-1; BOBO™-3; Bodipy492/515;Bodipy493/503; Bodipy500/510; Bodipy; 505/515; Bodipy 530/550; Bodipy542/563; Bodipy 558/568; Bodipy 564/570; Bodipy 576/589; Bodipy 581/591;Bodipy 630/650-X; Bodipy 650/665-X; Bodipy 665/676; Bodipy Fl; Bodipy FLATP; Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-Xconjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE;BO-PRO™-1; BO-PRO™-3; Brilliant Sulphoflavin FF; BTC; BTC-5N; Calcein;Calcein Blue; Calcium Crimson—; Calcium Green; Calcium Green-1 Ca²⁺ Dye;Calcium Green-2 Ca²⁺; Calcium Green-5N Ca²⁺; Calcium Green-C18 Ca²⁺;Calcium Orange; Calcofluor White; Carboxy-X-rhodamine (5-ROX); CascadeBlue™; Cascade Yellow; Catecholamine; CCF2 (GeneBlazer); CFDA; CFP (CyanFluorescent Protein); CFP/YFP FRET; Chlorophyll; Chromomycin A;Chromomycin A; CL-NERF; CMFDA; Coelenterazine; Coelenterazine cp;Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazinehcp; Coelenterazine ip; Coelenterazine n; Coelenterazine O; CoumarinPhalloidin; C-phycocyanine; CPM I Methylcoumarin; CTC; CTC Formazan;Cy2™; Cy3.1 8; Cy3.5™; Cy3™; Cy5.1 8; Cy5.5™; Cy5™; Cy7™; Cyan GFP;cyclic AMP Fluorosensor (FiCRhR); Dabcyl; Dansyl; Dansyl Amine; DansylCadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI;Dapoxyl; Dapoxyl 2; Dapoxyl 3′DCFDA; DCFH (DichlorodihydrofluoresceinDiacetate); DDAO; DHR (Dihydorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS(non-ratio); DiA (4-Di 16-ASP); Dichlorodihydrofluorescein Diacetate(DCFH); DiD-Lipophilic Tracer; DiD (DilC18(5)); DIDS; Dihydorhodamine123 (DHR); Dil (DilC18(3)); I Dinitrophenol; DiO (DiOC18(3)); DiR; DiR(DilC18(7)); DM-NERF (high pH); DNP; Dopamine; DsRed; DTAF; DY-630-NHS;DY-635-NHS; EBFP; ECFP; EGFP; ELF 97; Eosin; Erythrosin; Erythrosin ITC;Ethidium Bromide; Ethidium homodimer-1 (EthD-1); Euchrysin; EukoLight;Europium (111) chloride; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline);FIF (Formaldehyd Induced Fluorescence); FITC; Flazo Orange; Fluo-3;Fluo-4; Fluorescein (FITC); Fluorescein Diacetate; Fluoro-Emerald;Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1-43™; FM4-46; Fura Red™ (high pH); Fura Red™/Fluo-3; Fura-2; Fura-2/BCECF;Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink3G; Genacryl Yellow 5GF; GeneBlazer; (CCF2); GFP (S65T); GFP red shifted(rsGFP); GFP wild type' non-UV excitation (wtGFP); GFP wild type, UVexcitation (wtGFP); GFPuv; Gloxalic Acid; Granular blue;Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS;Hydroxycoumarin; Hydroxystilbamidine (FluoroGold); Hydroxytryptamine;Indo-1, high calcium; Indo-1 low calcium; Indodicarbocyanine (DiD);Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO JO-1; JO-PRO-1;LaserPro; Laurodan; LDS 751 (DNA); LDS 751 (RNA); Leucophor PAF;Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B;Calcein/Ethidium homodimer; LOLO-1; LO-PRO-1; Lucifer Yellow; LysoTracker Blue; Lyso Tracker Blue-White; Lyso Tracker Green; Lyso TrackerRed; Lyso Tracker Yellow; LysoSensor Blue; LysoSensor Green; LysoSensorYellow/Blue; Mag Green; Magdala Red (Phloxin B); Mag-Fura Red;Mag-Fura-2; Mag-Fura-5; Mag-lndo-1; Magnesium Green; Magnesium Orange;Malachite Green; Marina Blue; I Maxilon Brilliant Flavin 10 GFF; MaxilonBrilliant Flavin 8 GFF; Merocyanin; Methoxycoumarin; Mitotracker GreenFM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromobimane;Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green PyronineStilbene); NBD; NBD Amine; Neuro DiO; Nile Red; Nitrobenzoxedidole;Noradrenaline; Nuclear Fast Red; i Nuclear Yellow; Nylosan Brilliantlavin E8G; Oregon Green™; Oregon Green™ 488; Oregon Green™ 500; OregonGreen™ 514; Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5;PE-Cy7; PerCP; PerCP-Cy5.5; PE-TexasRed (Red 613); Phloxin B (MagdalaRed); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine3R; PhotoResist; Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26(Sigma); PKH67; PMIA; Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1;PO-I PRO-3; Primuline; Procion Yellow; Propidium lodid (P1); PyMPO;Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7;Quinacrine Mustard; Resorufin; RH 414; Rhod-2; Rhodamine; Rhodamine 110;Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green;Rhodamine Phallicidine; Rhodamine: Phalloidine; Rhodamine Red; RhodamineWT; Rose Bengal; R-phycocyanine; R-phycoerythrin (PE); rsGFP; S65A;S65C; S65L; S65T; Sapphire GFP; SBFI; Serotonin; Sevron Brilliant Red2B; Sevron Brilliant Red 4G; Sevron I Brilliant Red B; Sevron Orange;Sevron Yellow L; sgBFP™ (super glow BFP); sgGFP™ (super glow GFP); SITS(Primuline; Stilbene Isothiosulphonic Acid); SNAFL calcein; SNAFL-1;SNAFL-2; SNARF calcein; SNARF1; Sodium Green; SpectrumAqua;SpectrumGreen; SpectrumOrange; Spectrum Red; SPQ (6-methoxy-N-(3sulfopropyl) quinolinium); Stilbene; Sulphorhodamine B and C;Sulphorhodamine Extra; SYTO 11; SYTO 12; SYTO 13; SYTO 14; SYTO 15; SYTO16; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO 22; SYTO 23; SYTO 24; SYTO25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO 44; SYTO 45; SYTO 59; SYTO60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81; SYTO 82; SYTO83; SYTO 84; SYTO 85; SYTOX Blue; SYTOX Green; SYTOX Orange;Tetracycline; Tetramethylrhodamine (TRITC); Texas Red™; Texas Red-X™conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange;Thioflavin 5; Thioflavin S; Thioflavin TON; Thiolyte; Thiozole Orange;Tinopol CBS (Calcofluor White); TIER; TO-PRO-1; TO-PRO-3; TO-PRO-5;TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITCTetramethylRodaminelsoThioCyanate; True Blue; Tru Red; Ultralite;Uranine B; Uvitex SFC; wt GFP; WW 781; X-Rhodamine; XRITC; XyleneOrange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO 3; YOYO-1;YOYO-3; Sybr Green; Thiazole orange (interchelating dyes); semiconductornanoparticles such as quantum dots; or caged fluorophore (which can beactivated with light or other electromagnetic energy source), or acombination thereof.

A modifier unit such as a radionuclide can be incorporated into orattached directly to any of the compounds described herein byhalogenation. Examples of radionuclides useful in this embodimentinclude, but are not limited to, tritium, iodine-125, iodine-131,iodine-123, iodine-124, astatine-210, carbon-11, carbon-14, nitrogen-13,fluorine-18. In another aspect, the radionuclide can be attached to alinking group or bound by a chelating group, which is then attached tothe compound directly or by means of a linker. Examples of radionuclidesuseful in the apset include, but are not limited to, Tc-99m, Re-186,Ga-68, Re-188, Y-90, Sm-153, Bi-212, Cu-67, Cu-64, and Cu-62.Radiolabeling techniques such as these are routinely used in theradiopharmaceutical industry.

The radiolabeled compounds are useful as imaging agents to diagnoseneurological disease (e.g., a neurodegenerative disease) or a mentalcondition or to follow the progression or treatment of such a disease orcondition in a mammal (e.g., a human). The radiolabeled compoundsdescribed herein can be conveniently used in conjunction with imagingtechniques such as positron emission tomography (PET) or single photonemission computerized tomography (SPECT).

Labeling can be either direct or indirect. In direct labeling, thedetecting antibody (the antibody for the molecule of interest) ordetecting molecule (the molecule that can be bound by an antibody to themolecule of interest) include a label. Detection of the label indicatesthe presence of the detecting antibody or detecting molecule, which inturn indicates the presence of the molecule of interest or of anantibody to the molecule of interest, respectively. In indirectlabeling, an additional molecule or moiety is brought into contact with,or generated at the site of, the immunocomplex. For example, asignal-generating molecule or moiety such as an enzyme can be attachedto or associated with the detecting antibody or detecting molecule. Thesignal-generating molecule can then generate a detectable signal at thesite of the immunocomplex. For example, an enzyme, when supplied withsuitable substrate, can produce a visible or detectable product at thesite of the immunocomplex. ELISAs use this type of indirect labeling.

As another example of indirect labeling, an additional molecule (whichcan be referred to as a binding agent) that can bind to either themolecule of interest or to the antibody (primary antibody) to themolecule of interest, such as a second antibody to the primary antibody,can be contacted with the immunocomplex. The additional molecule canhave a label or signal-generating molecule or moiety. The additionalmolecule can be an antibody, which can thus be termed a secondaryantibody. Binding of a secondary antibody to the primary antibody canform a so-called sandwich with the first (or primary) antibody and themolecule of interest. The immune complexes can be contacted with thelabeled, secondary antibody under conditions effective and for a periodof time sufficient to allow the formation of secondary immune complexes.The secondary immune complexes can then be generally washed to removeany non-specifically bound labeled secondary antibodies, and theremaining label in the secondary immune complexes can then be detected.The additional molecule can also be or include one of a pair ofmolecules or moieties that can bind to each other, such as thebiotin/avadin pair. In this mode, the detecting antibody or detectingmolecule should include the other member of the pair.

Other modes of indirect labeling include the detection of primary immunecomplexes by a two step approach. For example, a molecule (which can bereferred to as a first binding agent), such as an antibody, that hasbinding affinity for the molecule of interest or corresponding antibodycan be used to form secondary immune complexes, as described above.After washing, the secondary immune complexes can be contacted withanother molecule (which can be referred to as a second binding agent)that has binding affinity for the first binding agent, again underconditions effective and for a period of time sufficient to allow theformation of immune complexes (thus forming tertiary immune complexes).The second binding agent can be linked to a detectable label orsignal-generating molecule or moiety, allowing detection of the tertiaryimmune complexes thus formed. This system can provide for signalamplification.

Immunoassays that involve the detection of as substance, such as aprotein or an antibody to a specific protein, include label-free assays,protein separation methods (i.e., electrophoresis), solid supportcapture assays, or in vivo detection. Label-free assays are generallydiagnostic means of determining the presence or absence of a specificprotein, or an antibody to a specific protein, in a sample. Proteinseparation methods are additionally useful for evaluating physicalproperties of the protein, such as size or net charge. Capture assaysare generally more useful for quantitatively evaluating theconcentration of a specific protein, or antibody to a specific protein,in a sample. Finally, in vivo detection is useful for evaluating thespatial expression patterns of the substance, i.e., where the substancecan be found in a subject, tissue or cell.

Provided that the concentrations are sufficient, the molecular complexes([Ab-Ag]n) generated by antibody-antigen interaction are visible to thenaked eye, but smaller amounts may also be detected and measured due totheir ability to scatter a beam of light. The formation of complexesindicates that both reactants are present, and in immunoprecipitationassays a constant concentration of a reagent antibody is used to measurespecific antigen ([Ab-Ag]n), and reagent antigens are used to detectspecific antibody ([Ab-Ag]n). If the reagent species is previouslycoated onto cells (as in hemagglutination assay) or very small particles(as in latex agglutination assay), “clumping” of the coated particles isvisible at much lower concentrations. A variety of assays based on theseelementary principles are in common use, including Ouchterlonyimmunodiffusion assay, rocket immunoelectrophoresis, andimmunoturbidometric and nephelometric assays. The main limitations ofsuch assays are restricted sensitivity (lower detection limits) incomparison to assays employing labels and, in some cases, the fact thatvery high concentrations of analyte can actually inhibit complexformation, necessitating safeguards that make the procedures morecomplex. Some of these Group 1 assays date right back to the discoveryof antibodies and none of them have an actual “label” (e.g. Ag-enz).Other kinds of immunoassays that are label free depend on immunosensors,and a variety of instruments that can directly detect antibody-antigeninteractions are now commercially available. Most depend on generatingan evanescent wave on a sensor surface with immobilized ligand, whichallows continuous monitoring of binding to the ligand. Immunosensorsallow the easy investigation of kinetic interactions and, with theadvent of lower-cost specialized instruments, may in the future findwide application in immunoanalysis.

The deposition of the nanoparticle-pharmaceutical agent complex can bedirect or indirect depending on the needs of the particular situation.For example, the nanoparticle-pharmaceutical agent complexes disclosedherein can be applied directly to the sclera of an eye. Also, by way ofexample, the nanoparticle-pharmaceutical agent complexes can be injectedinto the vitreal fluid whereby the complexes can then come into contactwith cells on the retina. One of skill in the art understands that theparticle method of applying the nanoparticle-pharmaceutical agentcomplex depends

In one aspect, the invention relates to a method of intracellulardelivery comprising administering an effective amount of a disclosednanoparticle to a subject. In a further aspect, the nanoparticle isfurther substituted with at least one biologically active agent, atleast one pharmaceutically active agent, and/or at least one imagingmoiety. In a further aspect, the method further comprises the step ofdegrading the nanoparticle.

In a further aspect, the invention relates to a method of intracellulardelivery comprising administering an effective amount of a disclosedpolymer or product of a disclosed method to a subject. In a furtheraspect, the polymer or product of a disclosed method is furthersubstituted with at least one biologically active agent, at least onepharmaceutically active agent, and/or at least one imaging moiety. In afurther aspect, the method further comprises the step of degrading thepolymer or product of a disclosed method.

K. Methods of Treatment

It is contemplated herein that degradable polyester nanoparticlesdisclosed herein will slowly release any agent encapsulated within thenanoparticle at a rate equivalent to the degradation of the particle.Such release over time is particularly useful for the time release ofpharmaceutical or biologically active agents that can be used to treatvarious diseases or conditions. Such diseases and conditions can includebut are not limited to ophthalmic disorders. Thus, disclosed herein aremethod of treating any of the ophthalmic disorder disclosed herein(e.g., glaucoma) comprising administering to a subject a degradablepolyester nanoparticle pharmaceutical or biologically active agentcomplex (nanoparticle complex).

The disclosed treatment methods may be used with any pharmaceutical orbiologically active agent known for use as a treatment for the givenophthalmic disorder to be treated. Thus, for example, the pharmaceuticalor biologically active agent can be an aptamer, an antibody, an alphaagonist, beta blocker, prostaglandin analog, carbonic anhydraseinhibitor, cholinergic, or any other agent disclosed herein.

Such agents are well known to those of skill in the art, but can includefor example, triamcinolone, ranibizumab, bevacizumab, pegaptanib(MACUGEN®), travoprost, bimatoprost, methazolamide, brinzolamide,Dorzolamide HCl, Acetazolamide, Timolol Maleate, Betaxolol HCl,Levobunolol HCl, Metipranolol, Timolol hemihydrate, Pilocarpine HCl,Carbachol, brimonidine tartrate, memantine, Apraclonidine HCl, orlatanoprost (XALATAN®). It is understood that the particular agent usedwill be suited to the medicinal purpose of the skilled artisan. Forexample, for treatment of macular degeneration, one or morepharmaceutical or biologically active agent such as ranibizumab orbevacizumab can be used. For the treatment of diabetic relateddisorders, triamcinolone can be used. For the treatment of glaucoma, oneor more agents such as pegaptanib (MACUGEN®), travoprost, bimatoprost,methazolamide, brinzolamide, Dorzolamide HCl, Acetazolamide, TimololMaleate, Betaxolol HCl, Levobunolol HCl, Metipranolol, Timololhemihydrate, Pilocarpine HCl, Carbachol, brimonidine tartrate,Apraclonidine HCl, memantine, or latanoprost (XALATAN®) can be used inthe disclosed methods. Thus, disclosed herein are methods of treating anophthalmic disorder comprising administering to a subject one or more ofthe pharmaceutical or biologically active agents selected from the groupconsisting of triamcinolone, ranibizumab, bevacizumab, pegaptanib(MACUGEN®), travoprost, bimatoprost, methazolamide, brinzolamide,Dorzolamide HCl, Acetazolamide, Timolol Maleate, Betaxolol HCl,Levobunolol HCl, Metipranolol, Timolol hemihydrate, Pilocarpine HCl,memantine, Carbachol, brimonidine tartrate, Apraclonidine HCl, andlatanoprost (XALATAN®).

It is disclosed herein that the treatment of any of the ophthalmicdisorders disclosed herein may be treated by the use of more than onepharmaceutical or biologically active agent used in combination in thenanoparticle complexes. For example, disclosed herein are methods oftreating glaucoma comprising administering to a subject a degradablepolyester nanoparticle pharmaceutical agent complex wherein the complexcomprises at least two pharmaceutical agents. It is understood that thedisclosed methods of treatment or modulating receptor or enzymatictreatment can utilize within the nanoparticle complex any comprising acombination of at least two or more pharmaceutical or biologicallyactive agents disclosed herein. For example, a combination ofpharmaceutical agents may comprise an alpha agonist and a beta blockersuch as Brimonidine Tartrate and Timolol Maleate or a beta blocker and acarbonic anhydrase inhibitor such as Dorzolomide HCl and TimololMaleate. Other combinations contemplated herein include two or morealpha agonists, two or more beta blockers, two or more cholinergics, twoor more carbonic anhydrases, two or more prostaglandin analogs, two ormore antibodies, an alpha agonist and a beta blocker, an alpha agonistand a carbonic anhydrase inhibitor, an alpha agonist and a cholinergic,an alpha agonist and a carbonic anhydrase inhibitor, an alpha agonistand a prostaglandin analog, an alpha agonist an antibody, a beta blockerand a carbonic anhydrase inhibitor, a beta blocker and a prostaglandinanalog, a beta blocker and an antibody, a beta blocker an a cholinergic,a carbonic anhydrase inhibitor and a prostaglandin analog, a carbonicanhydrase inhibitor and a cholinergic, a carbonic anhydrase inhibitorand an antibody, a cholinergic and a prostaglandin analog, a cholinergicand an antibody, and a prostaglandin analog and an antibody.

The mechanism by which the agents for use in the disclosed nanoparticleshave their effect are known to those of skill in the art. For example,those of skill in the art know that the alpha agonist disclosed hereinsuch as brimonidine tartrate and Apraclonidine HCl, function byinteracting with a G coupled protein receptor known as the alphaadrenergic receptor. Similarly, the mechanism by which beta blockersfunction is two inhibit the functioning of a G coupled protein receptorreferred to as the beta adrenergic receptor. Inhibitors of the betaadrenergic receptor include but are not limited to Timolol Maleate,Betaxolol HCl, Levobunolol HCl, Metipranolol, and Timolol hemihydrate.It is further understood that some agents which act as modulators of Gcoupled protein receptors are analogs to the natural ligand for thereceptor. For example, latanoprost, travoprost, and bimatoprost areprostaglandin receptor analogs which modulate the activity of theprostaglandin F2 (FP) receptor. The activity of other receptors such asthe acetylcholine receptor can also be modulated by the activity of theagents disclosed herein. For example, Pilocarpine HCl or Carbacholmodulate acetylcholine receptor activity. Thus, disclosed herein aremethods of modulating a receptor on a cell comprising contacting thereceptor with a degradable polyester nanoparticle pharmaceuticalbiologically active agent complex (nanoparticle complex), wherein one ormore pharmaceutical or biologically active agents is encapsulated by adegradable polyester nanoparticle. Also disclosed are methods ofmodulating a receptor wherein the receptor is a G coupled proteinreceptor such as the alpha adrenergic receptor, the beta adrenergicreceptor, or prostaglandin F2 (FP) receptor comprising administering toa subject the nanoparticle complexes disclosed herein. Additionally,disclosed are methods of modulating a receptor wherein the receptor isthe acetylcholine receptor comprising administering to a subject thenanoparticle complexes disclosed herein.

It is further contemplated herein that not all of the agents disclosedherein are known to those of skill in the art for use in the methods oftreatment function by modulating the activity of a receptor. Some of theagents described herein have their medicinal effect through the abilityto change the effect of an enzyme. For example, VEGF and in particularVEGF-A effects the outflow of vitreal fluid. Agents such astriamcinolone (a steroid) or pegaptanib (an aptamer) bind and inhibitVEGF whereas ranibizumab or bevacizumab are antibodies with a morespecific action of inhibiting VEGF-A. Other agents such asmethazolamide, brinzolamide, Dorzolamide HCl, and Acetazolamide inhibitcarbonic anhydrase. Therefore disclosed herein are methods of modulatingthe activity of an enzyme such as VEGF, VEGF-A, or carbonic anhydrasecomprising administering to a subject comprising administering to asubject the nanoparticle complexes disclosed herein. Due to the effectof enzyme activity on viteous outflow or other biological activityassociated with ophthalmic disorders, disclosed herein are methods oftreating an ophthalmic disorder (e.g., glaucoma, macular degeneration ordiabetic odema) comprising administering to a subject a nanoparticlecompex, wherein one or more pharmaceutical or biologically active agentencapsulated by the nanoparticle modulates that activity of VEGF,VEGF-A, or carbonic anhydrase.

L. Uses

Also provided are uses of the disclosed polymers, nanoparticles, andproducts. In one aspect, the invention relates to a use of a disclosedpolymer or a disclosed nanoparticle to deliver a biologically activeagent, a pharmaceutically active agent, and/or an imaging moiety. Thedisclosed compounds, compositions, and conjugates and practicalsynthesis of same provide approaches for applications in cancertreatment and drug delivery across biological barriers such as thecornea, tissues, skin, and the blood brain barrier.

These degradable polymers find application in controlled releasetechnologies that have to penetrate tissues and cellular membranes.Thus, the nanoparticle-dendrimer conjugates comprising a discloseddegradable nanoparticle and a disclosed intracellular deliverycomposition can hold and deliver therapeutics ranging from smallmolecules to larger peptides, proteins, and antibodies.

In a further aspect, the invention relates to a use of a disclosedpolymer or a disclosed nanoparticle for trancomeal delivery of abiologically active agent, a pharmaceutically active agent, and/or animaging moiety.

Many regions of the eye are relatively inaccessible to systemicallyadministered agents. For example, orally administered agents passthrough the liver before reaching estrogen sensitive tissues. Becausethe liver contains enzymes that can inactivate the agent, the agent thateventually reaches tissue targeted for treatment can be virtuallyineffective. Moreover, systemic administration risks production ofundesirable side effects. It can also be problematic to deliver abiologically active agent, a pharmaceutically active agent, and/or animaging moiety into the eye via invasive procedures such as injection.Further still, patient compliance can be low in cases of invasiveadministration.

As a result, topical drug delivery remains the preferred route ofadministration to the eye. There are a variety of factors that affectthe absorption of drugs into the eye. These factors include: theinstillation volume of the drug, the frequency of instilled drugadministration, the structure and integrity of the cornea, the proteinlevel in tears, the level of enzymes in tears, lacrimal drainage andtear turnover rate, as well the rate of adsorption and absorption of adrug by the conjunctiva, sclera, and eyelids. A potential way ofreducing or even eliminating systemic side effects is to improve oculartargeting that would allow for the use of reduced doses of thebiologically active agent in the ophthalmic drug formation.

A major barrier to ocular drug penetration is the cornea. The cornea iscomposed of three layers: a lipid-rich epithelium, a lipid-poor soma,and a lipid-rich endothelium. Therefore, an agent must possess bothlipophilic-hydrophilic balance for adequate transcorneal penetrationand, thus, ocular bioavailability (Akers, H. J., “Ocular bioavailabilityof topically applied ophthalmic drugs,” Am Pharm, NS23:33-36 (1983)).

Thus, in one aspect, the disclosed compounds provide improvedphysicochemical properties including, but not limited to, favorableocular bioavailability and facile transcomeal penetration.

In another aspect, the disclosed compounds treat and/or protect againstvarious ocular diseases. That is, the disclosed compounds can be used todiagnose, prevent, and/or treat ophthalmic disorders. Preferreddisclosed compounds can be effective in treating and/or preventingmaladies associated with vision-threatening intraocular damage due topathophysiological predispositions. Preferred disclosed compoundsinclude those which treat retinal infection, glaucoma, and/or maculardegeneration.

M. Manufacture of a Medicament

Also provided is a method for the manufacture of a medicament. In oneaspect, the invention relates to a method for the manufacture of amedicament for delivery of a biologically active agent, apharmaceutically active agent, and/or an imaging moiety comprisingcombining at least one disclosed polymer or at least one disclosednanoparticle with a pharmaceutically acceptable carrier.

In a further aspect, the pharmaceutical composition relates to acomposition for preventing and/or treating ophthalmic disorders.

N. Pharmaceutical Compositions

In one aspect, the invention relates to pharmaceutical compositionscomprising the disclosed compositions. That is, a pharmaceuticalcomposition can be provided comprising a therapeutically effectiveamount of one or more disclosed polymer and/or one or more products of adisclosed method and/or one or more disclosed nanoparticle and apharmaceutically acceptable carrier for administration in a mammal. In afurther aspect, the one or more disclosed polymer and/or one or moreproducts of a disclosed method and/or the one or more disclosednanoparticle is further substituted with at least one biologicallyactive agent, at least one pharmaceutically active agent, and/or atleast one imaging moiety.

The disclosed pharmaceutical compositions can further comprise othertherapeutically active compounds, which are usually applied in thetreatment of the above mentioned pathological conditions. It isunderstood that the disclosed compositions can be employed in thedisclosed methods of using.

O. Kits

Also provided are kits related to the disclosed compositions. In oneaspect, the invention relates to a kit comprising at least one disclosedpolymer, at least one disclosed nanoparticle or at least one product ofa disclosed method. It is understood that the disclosed kits can be usedin connection with the disclosed methods of using.

Thus, in one aspect, the invention related to kits comprising a firstdegradable polyester nanoparticle and a first biologically active agent,first pharmaceutically active agent, or first imaging agent encapsulatedwithin the first nanoparticle, and one or more of: a second biologicallyactive agent, second pharmaceutically active agent, or second imagingagent encapsulated within the first nanoparticle, wherein the firstbiologically active agent, first pharmaceutically active agent, or firstimaging agent is different from the second biologically active agent,second pharmaceutically active agent, or second imaging agent; or asecond degradable polyester nanoparticle and a second biologicallyactive agent, second pharmaceutically active agent, or second imagingagent encapsulated within the second nanoparticle, wherein the firstbiologically active agent, first pharmaceutically active agent, or firstimaging agent is different from the second biologically active agent,second pharmaceutically active agent, or second imaging agent; apharmaceutically acceptable carrier; or instructions for treating adisorder known to be treatable by the first biologically active agent orfirst pharmaceutically active agent.

In a further aspect, at least one agent is brominidine tartrate. In afurther aspect, at least one agent is an inhibitor of VEGF. In a furtheraspect, at least one agent is an inhibitor of VEGF-A. In a furtheraspect, at least one agent is a alpha agonist, beta blocker,prostaglandin analog, carbonic anhydrase inhibitor, antibody, aptamer,or cholinergic. In a further aspect, at least one agent is selected fromtriamcinolone, ranibizumab, bevacizumab, pegaptanib (MACUGEN®),travoprost, bimatoprost, methazolamide, brinzolamide, dorzolamide HCl,acetazolamide, memantine, timolol maleate, betaxolol HCl, levobunololHCl, metipranolol, timolol hemihydrate, pilocarpine HCl, carbachol,brimonidine tartrate, apraclonidine HCl, and latanoprost (XALATAN®).

P. Experimental

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary of theinvention and are not intended to limit the scope of what the inventorsregard as their invention. Efforts have been made to ensure accuracywith respect to numbers (e.g., amounts, temperature, etc.), but someerrors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in ° C. or is atambient temperature, and pressure is at or near atmospheric.

1. Characterization Methods

¹H NMR spectra were obtained from a Bruker AC300 Fourier TransformSpectrometer, with CDCl₃ in TMS as the solvent. ¹³C NMR spectra wereobtained from a Bruker AC400 Fourier Transform Spectrometer with CDCl₃as the solvent.

Gel-permeation chromatography (GPC) was performed on a Waterschromatograph equipped with a Waters 2414 refractive index detector, aWaters 2481 dual λ absorbance detector, a Waters 1525 binary HPLC pump,and four 5 mm Waters columns (300 mm×7.7 mm), connected in series withincreasing pore size (100, 1000, 100,000 and 1,000,000 {acute over (Å)}respectively). All runs were performed with tetrahydrofuran (THF) as theeluent at a flow rate of 1 mL/min.

For dynamic light scattering (DLS) a Zetasizer Nano Series instrumentwith a CGS-3 compact goniometer system by Malvern Instruments (MalvernZetasizer Nanoseries, Malvern, UK) was employed at a fixed angle of 90°at 25° C., taking the average of three measurements. The particles werediluted with toluene to a concentration of 5-6 mg/mL, which gave thedesired number of counts in order to obtain a good signal-to-noiseratio.

Samples for transmission electron microscopy (TEM) imaging were preparedby dissolving 0.5 mg nanoparticles in 1 mL isopropanol and 0.3 mLacetonitrile. The samples were sonicated for 5 min and were stained with2 drops of 3% phosphotungstic acid. The carbon grids were prepared byplacing a drop of dispersed particles onto an Ultrathin Carbon Type-A400 Mesh Copper Grid (Ted Pella, Inc., Redding, Calif.) and drying atambient temperature. A Philips CM20T transmission electron microscopeoperating at 200 kV in bright-field mode was used to obtain TEMmicrographs of the polymeric nanoparticles.

Samples were centrifuged at 600 rpm on a Model CS InternationalCentrifuge from International Equipment Company (Boston, Mass.).

2. Materials

Reagent chemicals were purchased from Aldrich (Milwaukee, Wis.), EMD,Alfa-Aesar, Fisher Scientific, and Acros and used as received, unlessotherwise stated. Spectra/Por® Dialysis membrane and SnakeSkin® PleatedDialysis Tubing, regenerated cellulose, were purchased from SpectrumLaboratories Inc. and Pierce Biotechnology, respectively. Analytical TLCwas performed on commercial Merck plates coated with silica gel GF254(0.24 mm thick). Silica gel for flash chromatography was Merck Kieselgel60 (230-400 mesh, ASTM) or Sorbent Technologies 60 Å (40-63 μm,technical grade). MAL-dPeg™₄-t-boc-hydrazide was obtained from QuantaBiodesign, Ltd. (Powell, Ohio) and used as received. Cy3 NHS dye andPD-10 Desalting columns were received from GE Healthcare (Piscataway,N.J.). Spectra/Por® Biotech Cellulose Ester (CE) Dialysis Membranes(1,000 MWCO) obtained from Spectrum Laboratories, Inc. (RanchoDominguez, Calif.). SnakeSkin® Pleated Dialysis Tubing (10,000 MWCO) wasobtained from Pierce Biotechnology, Inc. (Rockford, Ill.). Absolutemolecular weight was determined with static light scattering.

3. Synthesis of A-Allyl-Δ-Valerolactone (avl) (B)

A 500 mL round bottom flask, equipped with stir bar, was sealed with aseptum, purged with nitrogen for 30 min and cooled in a dry ice/acetonebath. A solution of lithium diisopropylamine (2.0 M in THF/heptane/ethylbenzene, 33 mL, 66 mmol) was added to the round bottom flask. A nitrogenpurged solution of 6-valerolactone (5.43 mL, 60 mmol) in THF (60 mL) wasadded dropwise via syringe over 1.5 h. After an additional 30 min ofstirring, a solution of allyl bromide (6.21 mL, 72 mmol) inhexamethylphosphoramide (12.51 mL, 72 mmol) was added dropwise viasyringe over 30 min. The reaction mixture was warmed up to −40° C. usinga dry ice/acetone bath and stirred for 3 h. The reaction was quenchedwith excess NH₄Cl solution and warmed to room temperature. The crudeproduct was washed twice with brine, dried with anhydrous magnesiumsulfate and concentrated via rotary evaporator. Column chromatographyusing CH₂Cl₂ gave a viscous yellow product. Yield: 3.4262 g (41%). ¹HNMR (300 MHz, CDCl₃/TMS, ppm) δ: 5.7 (m, 1H, H₂C═CH—), 5.08 (m, 2H,H₂C═CH—), 4.28 (m, 2H, —C(O)OCH₂—), 2.53-2.58 (m, 2H, H₂C═CHCH₂—), 2.27(m, 1H, H₂C═CHCH₂CH—), 2.06 (m, 1H, H₂C═CHCH₂CHCH₂—), 1.89 (m, 2H,C(O)OCH₂CH₂—), 1.55 (m, 1H, H₂C═CHCH₂CHCH₂—); ¹³C NMR (400 MHz, CDCl₃,ppm) δ: 173.8 (—C(O)O—), 135.0 (H₂C═CH—), 117.4 (H₂C═CH—), 68.4(—C(O)OCH₂—), 39.2 (H₂C═CHCH₂CH—), 35.4 (H₂C═CHCH₂—), 24.0(—CH₂CH₂CH₂—), 21.9 (—CH₂CH₂CH₂—).

4. Synthesis of Copolymer poly(vl-avl) (Ab)

A 50 mL 3-necked round bottom flask, equipped with stir bar, was sealedwith two septa and a gas inlet. The flask was evacuated and refilledwith nitrogen three times. Stock solutions of 1.7 M ethanol (EtOH) inTHF and 3.7×10⁻² M tin(II) 2-ethylhexanoate (Sn(Oct)₂) in THF were madein sealed N₂ purged flasks. Solutions of EtOH (0.32 mL, 5.410×10⁻¹ mmol)and Sn(Oct)₂ (0.30 mL, 1.12×10⁻² mmol) were combined in the nitrogenpurged 50 mL flask. After stirring the mixture for 30 min,α-allyl-δ-valerolactone (1.16 g, 8.32 mmol) and 6-valerolactone (vl, 2.5g, 24.97 mmol) were added. The reaction vessel stirred in a 105° C. oilbath for 48 h. Residual monomer and catalyst were removed by dialyzingwith Spectra/Por® dialysis membrane (MWCO=1000) against CH₂Cl₂ to give agolden brown polymer. Yield: 3.2398 g (88%). M_(w)=4834 Da, PDI=1.17; ¹HNMR (300 MHz, CDCl₃/TMS, ppm) δ: 5.7 (m, H₂C═CH—), 5.09 (m, H₂C═CH—),4.09 (m, —CH₂—O—), 3.65 (m, CH₃CH₂O—), 2.35 (m, vl, —CH₂CH₂C(O)O—, avl,H₂C═CHCH₂CH—, H₂C═CHCH₂CH—), 1.68 (m, avl & vl, —CHCH₂CH₂—), 1.25 (t,CH₃CH₂O—); ¹³C NMR (400 MHz, CDCl₃, ppm) 8:174.6 (avl, —C(O)—), 172.7(vl, —C(O)—), 134.6 (H₂C═CH—), 116.4 (H₂C═CH—), 63.3, 44.3, 35.9, 33.1,27.5, 25.9, 23.6, 20.9.

5. Synthesis of A-Propargyl-Δ-Valerolactone (pvl) (C)

A 250 mL round bottom flask, equipped with stir bar, was sealed with aseptum, purged with nitrogen for 30 min and cooled in a dry ice/acetonebath. A solution of lithium diisopropylamine (2.0 M in THF/heptane/ethylbenzene, 22 mL, 44 mmol) was added to the flask. A nitrogen purgedsolution of 6-valerolactone (3.62 mL, 40 mmol) in THF (40 mL) was addeddropwise via syringe over 1.5 h. After an additional 30 min of stirring,a solution of propargyl bromide (4.34 mL, 48 mmol) inhexamethylphosphoramide (8.4 mL, 48 mmol) was added dropwise via syringeover 20 min. The reaction mixture was warmed up to −30° C. using a dryice/acetone bath and stirred for 3 h. The reaction was quenched withexcess NH₄Cl solution and warmed to room temperature. The crude productwas washed twice with brine, dried with anhydrous magnesium sulfate andconcentrated via rotary evaporator. Column chromatography with CH₂Cl₂gave a viscous yellow product. Yield: 2.8194 g (50.6%). ¹H NMR (300 MHz,CDCl₃/TMS, ppm) δ: 4.35 (m, 2H, —C(O)OCH₂—), 2.69 (m, 2H,—C(O)CHCH₂C≡CH), 2.53 (m, 1H —C(O)CHCH₂C≡CH), 2.29 (m, 1H, —CHCH₂CH₂—),2.05 (s, 1H, HC≡CCH₂—), 1.96 (m, 2H, —CHCH₂CH₂—), 1.74 (m, 1H,—CHCH₂CH₂—); ¹³C NMR (400 MHz, CDCl₃, ppm) δ: 172.8, 80.8, 70.1, 68.5,38.5, 23.8, 21.7, 20.4.

6. Synthesis of Copolymer Poly (vl-avl-pvl) (AbC)

A 50 mL 3-necked round-bottom flask, equipped with stir bar, was sealedwith two septa and a gas inlet. The flask was evacuated and refilledwith nitrogen three times. Stock solutions of 1.7 M ethanol in THF and3.7×10⁻² M Sn(Oct)₂ in THF were made in sealed N₂ purged flasks.Solutions of ethanol (0.21 mL, 3.69×10⁻¹ mmol) and Sn(Oct)₂ (0.20 mL,5.41×10⁻³ mmol) were combined in the nitrogen purged 50 mL flask. Afterstirring the mixture for 30 min, α-allyl-δ-valerolactone (0.8 g, 5.7mmol), δ-valerolactone (1.26 g, 12.6 mmol) andα-propargyl-δ-valerolactone (0.63 g, 4.6 mmol) were added. The reactionvessel stirred in a 105° C. oil bath for 48 h. Residual monomer andcatalyst were removed by dialyzing with Spectra/Por® dialysis membrane(MWCO=1000) against CH₂Cl₂ to give a golden brown polymer. Yield: 2.25 g(84%). M_(w)=3500 Da, PDI=1.26; ¹H NMR (300 MHz, CDCl₃/TMS, ppm) δ: 5.71(m, H₂C═CH—), 5.03 (m, H₂C═CH—), 4.08 (m, —CH₂O—), 3.65 (m, CH₃CH₂O—),2.55 (m, pvl, —C(O)CH—, —CHCH₂C≡CH), 2.45 (m, —CH₂C≡CH), 2.34 (m, vl,—CH₂CH₂C(O)O—, avl, H₂C═CHCH₂CH—, H₂C═CHCH₂CH—), 2.02 (m, pvl, —C≡CH),1.68 (m, pvl, avl & vl, —CHCH₂CH₂—), 1.259 (t, CH₃CH₂O—); ¹³C NMR (400MHz, CDCl₃, ppm) δ: 174.6, 172.7, 133.6, 117.2, 80.7, 69.9, 63.3, 44.3,35.9, 33.1, 27.5, 25.9, 23.6, 20.9.

7. Synthesis of 2-Oxepane-1,5-Dione (opd) (D)

A 100 mL round bottom flask, equipped with stir bar, was charged with1,4-cyclohexanedione (2.0 g, 17.84 mmol) and 3-chloroperoxybenzoic acid(4.5 g, 26.08 mmol). Dichloromethane (22 mL) was added and the reactionmixture stirred and refluxed for 3 h at 40° C. The reaction mixture wascooled to room temperature and dried with anhydrous MgSO₄. Solvent wasremoved via rotary evaporation. The crude product was washed three timeswith cold diethyl ether (100 mL for each wash) and dried in vacuo atroom temperature. Yield: 1.4814 g (64.7%). ¹H NMR (300 MHz, CDCl₃/TMS,ppm) δ: 4.4 (t, 2H, —C(O)OCH₂CH₂C(O)—), 2.84 (dd, 2H, —CH₂C(O)O—), 2.72(m, 4H, —CH₂C(O)CH₂—); ¹³C NMR (400 MHz, CDCl₃, ppm) δ: 204.9 (—C(O)—),173.3 (—C(O)O—), 63.3 (—CH₂O—), 44.7 (—OCH₂CH₂C(O)—), 38.6(—C(O)CH₂CH₂C(O)—), 27.9 (—CH₂C(O)O—).

8. Synthesis of Copolymer poly(vl-avl-opd) (AbD)

To a 50 mL 3-necked round bottom flask, equipped with stir bar,condenser, nitrogen purge and septa, 2-oxepane-1,5-dione (0.6987 g, 5.45mmol) and dry toluene (4 mL) was added. The mixture stirred in an oilbath at 70° C. to dissolve the monomer. Upon dissolving, δ-valerolactone(1.5 g, 14.98 mmol), α-allyl-δ-valerolactone (0.9546 g, 6.81 mmol),absolute ethanol (0.0205 g, 4.4×10¹ mmol) and Sn(Oct)₂ (0.0119 g,2.73×10⁻² mmol) were then added to the reactor and the mixture washeated for 48 h at 110° C. Residual monomer and catalyst were removed bydialyzing with Spectra/Por® dialysis membrane (MWCO=1000) against CH₂Cl₂to give a golden brown polymer. Yield: 2.6894 g (85%). M_(w)=4858 Da,PDI=1.27; ¹H NMR (300 MHz, CDCl₃/TMS, ppm) δ: 5.72 (m, H₂C═CH—), 5.06(m, H₂C═CH—), 4.34 (m, —CH₂CH₂C(O)CH₂CH₂O—), 4.08 (m, —CH₂O—), 3.67 (m,—OCH₂CH₃), 2.78 (m, opd, —OC(O)CH₂CH₂C(O)CH₂—), 2.58 (m, opd,—OC(O)CH₂CH₂C(O)CH₂—), 2.34 (m, vl, —CH₂CH₂C(O)O—, avl, H₂C═CHCH₂CH—,H₂C═CHCH₂CH—), 1.66 (m, avl & vl, —CHCH₂CH₂—), 1.25 (t, —CH₂CH₃); ¹³CNMR (400 MHz, CDCl₃, ppm) δ: 204.9, 175.2, 173.7, 173.2, 135.0, 117.0,63.9, 44.8, 36.4, 33.6, 28.0, 26.3, 21.3.

9. Synthesis of Copolymer poly(vl-avl-pvl-opd) (AbCD)

To a 25 mL 3-necked round bottom flask, equipped with stir bar,2-oxepane-1,5-dione (0.2626 g, 2.05 mmol) was added and the flask wassealed with two septa and a gas inlet. The flask was evacuated andrefilled with argon three times. Dry toluene (1.25 mL) was added and themixture stirred in an oil bath at 70° C. to dissolve the monomer. Upondissolving, Sn(Oct)₂ (0.0018 g, 4.41×10⁻³ mmol in 0.15 mL dry toluene),absolute ethanol (12.8 μL, 2.22×10⁻¹ mmol), δ-valerolactone (0.62 g, 6.2mmol), α-allyl-δ-valerolactone (0.38 g, 2.69 mmol), andα-propargyl-δ-valerolactone (0.38 g, 2.73 mmol) were added. Thetemperature of the oil bath was increased to 105° C. and the mixturestirred for 50 h. Residual monomer and catalyst were removed bydialyzing with Spectra/Por® dialysis membrane (MWCO=1000) against CH₂Cl₂to give a golden brown polymer. Yield: 1.31 g (80%). M_(w)=3525 Da,PDI=1.27; ¹H NMR (300 MHz, CDCl₃/TMS, ppm) δ: 5.86 (m, H₂C═CH—), 5.09(m, H₂C═CH—), 4.34 (m, opd, —CH₂CH₂C(O)CH₂CH₂O—), 4.08 (m, avl, pvl &vl, —CH₂O—), 3.65 (m, —OCH₂CH₃), 2.74 (m, opd, —OC(O)CH₂CH₂C(O)—), 2.60(m, opd, —CH₂CH₂C(O)CH₂CH₂—, pvl, —OC(O)CH—, —CHCH₂C≡CH), 2.50 (m,CHCH₂C≡CH), 2.34 (m, vl, —CH₂CH₂C(O)O—, avl, H₂C═CHCH₂CH—,H₂C═CHCH₂CH—), 2.02 (m, HC≡C—), 1.68 (m, pvl, avl & vl, —CHCH₂CH₂—),1.25 (m, —CH₂CH₃).

10. General Procedure for Oxidation of Copolymers

In a 200 mL round bottom flask, equipped with stir bar, poly(vl-avl)(2.7389 g, 6.12 mmol) was dissolved in 37 mL of CH₂Cl₂. To thissolution, 3-chloroperoxybenzoic acid (2.0903 g, 12.11 mmol) was addedslowly. The mixture was stirred for 72 h at room temperature and thenconcentrated via rotary evaporator. The crude product was dissolved in aminimal amount of THF (5 mL) and poured into a round-bottomed flaskcontaining 1 L diethyl ether. The solution was kept overnight at 0° C.and a white solid was obtained. The solution was decanted off and thesolid was dried in vacuo to obtain poly(vl-evl). Yield: 1.9467 g (71%).¹H NMR (300 MHz, CDCl₃/TMS, ppm) δ: The significant change is thedisappearance of the allylic protons at 5.7 and 5.09 ppm and theappearance of small broad resonance peaks at 2.96, 2.75 and 2.47 ppm dueto the formation of the epoxide ring. All other aspects of the spectrumare similar.

11. General Procedure for Nanoparticle Formation

In a 100 mL three-necked round bottom flask equipped with stir bar,condenser and septa, a solution of 2,2′-(ethylenedioxy)diethylamine(39.3 μL, 2.68×10⁻⁴ mol) in 27.6 mL CH₂Cl₂. A solution of poly(vl-evl)(0.1330 g, M_(w)=4834 Da, PDI=1.17) dissolved in CH₂Cl₂ (0.18 mL) wasadded dropwise via a peristaltic pump at 13 mL/min with vigorousstirring. The mixture was heated at reflux for a total of 12 h. Residualdiamine was removed by dialyzing with SnakeSkin® Pleated Dialysis Tubing(MWCO=10,000) against dichloromethane. ¹H NMR (300 MHz, CDCl₃/TMS, ppm)δ: The significant change is the disappearance of the epoxide protons at2.96, 2.75 and 2.47 ppm and the appearance of signals at 3.5 and 2.89ppm corresponding to the protons neighboring the secondary amine of thePEG linker after cross-linking. All other aspects of the spectrum aresimilar.

12. Determination of Amine Content

Nanoparticles can be titrated with a strong acid to determine aminecontent. As shown in Table 2, several poly(vl-evl) (AB) nanoparticlesamples were titrated with perchloric acid to determine the weightpercentages (wt %) of primary amine and secondary amine in the threesamples that we analyzed with transmission electron microscopy. Thethree samples (shown in Table 2) titrated have the following sizedimensions by DLS: 58.06, 255.7 and 425.1 nm.

TABLE 2 Correlation of particle size and amine content AB Nanoparticlesize (nm) Primary amine wt % Secondary amine wt % 58.06 0.008% 0.031%255.7 0.025% 0.098% 425.1 0.055%  0.20%

13. Nanoparticles Formed by Co-Polymerization

While nanoparticles are typically prepared with a single type of polymeror copolymer, nanoparticles have also been successfully produced from amixture of poly(vl-evl-pvl) and poly(vl-evl-opd). Such nanoparticles aretabulated in Table 3.

TABLE 3 Nanoparticles formed from two polymers Diameter (nm)Poly(vl-evl-pvl) with Amine/1 Epoxide poly(vl-evl-opd) 4  43.7 ± 4.50 894.15 ± 6.85

14. Varying Comonomer Content

The properties of nanoparticles can be further tailored by incorporatingdifferent percentages of epoxy-δ-valerolactone (evl) into the polymerbackbone. The data summarized in Table 4, below, shows the nanoparticlesmade from the linear poly(vl-evl) with 2% evl, 7% evl, and 19% evl.These data show that, as the % evl is decreased to 2% in the linearpolymer, smaller nanoparticles can be obtained. As the % evl is increaseto 19%, the resulting nanoparticles are larger but have a smalldeviation in comparison to the larger nanoparticles made frompoly(vl-evl) with 7% evl.

TABLE 4 Effect of varying comonomer content Diameter (nm) Diameter (nm)Diameter (nm) Poly(vl-evl) Poly(vl-evl) Poly(vl-evl) 2% evl 7% evl 19%evl Amine/1 Epoxide AB 3  7.02 ± 1.05  82.1 ± 5.73 179.9 ± 18.0 4 19.04± 1.32 115.6 ± 25.4 225.6 ± 22.5 5 33.55 ± 1.93 255.7 ± 60.3 299.0 ±31.2 6 48.66 ± 3.18 342.2 ± 52.2 409.1 ± 42.7 8  84.89 ± 10.47 425.1 ±100  843.3 ± 88.0

The relationship between reaction stoichiometry and particle size forvarying comonomer content is further illustrated in FIG. 17-FIG. 19.

15. Addition of Ethylenediamine 2-Vinylsulfonyl-Ethyl Carbonate to ABD(poly(vl-evl-opd)) Nanoparticles

In a 100 mL round bottom flask, equipped with stir bar, ABDnanoparticles (0.0846 g, 2.45×10⁻⁴ mmol) were dissolved in 12.5 mL ofCH₂Cl₂. To this solution, ethylenediamine 2-(vinylsulfonyl)-ethylcarbonate in methanol (0.0152 g in 69 μL methanol, 5.89×10⁻² mmol) wasadded. Sodium cyanoborohydride (0.0111 g, 1.76×10⁻¹ mmol) was dissolvedin 12.5 mL methanol and added to the round bottom flask. The pH of thereaction mixture was adjusted to a pH of 6.5 with aqueous 1 M NaOH and 1M HCl. The mixture was stirred for 25 h at room temperature and thendialyzed with SnakeSkin® Pleated Dialysis Tubing (MWCO=10,000) against1:1 dichloromethane/methanol. Successful attachment of the linker wasobserved by the appearance of signals 6.7 ppm and 6.9 ppm (¹H NMR, 300MHz, CDCl₃/TMS) due to the vinyl protons of the linker.

16. Attachment of GV-13-Alexafluor 750 to ABD Nanoparticles

In a small vial, equipped with a stir bar, linker modified nanoparticles(L-ABD) (29.9 mg) were dissolved in 800 μL PBS buffer (pH 7.2) and 700μL dimethylformamide. To this solution, 251 μL GV-13-Alexafluor (0.44 mgin 150 μL PBS buffer and 26.5 μL DMF) was added to the vial viamicropipette. After 45 min of stirring at room temperature, GV-13 (2.08mg, 1.9×10⁻³ mmol) dissolved in 200 μL PBS buffer was added. Thereaction mixture stirred for 24 h in aluminum covered beaker. Theresulting mixture was purified with concentrating tubes (MWCO=10,000) toremove excess GV-13 and GV-13-Alexafluor. The purified product wasconcentrated via rotary evaporator. Successful attachment of peptide anddye was observed by the presence of a bright blue color due to the dye.¹H NMR also shows the presence of the peptide.

General.

Commercial reagents were obtained from commercial sources (Aldrich, EMD,Alfa-Aesar, Fisher Scientific, and Acros) and used without furtherpurification. Analytical TLC was performed on commercial Merck platescoated with silica gel GF254 (0.24 mm thick) and spots located by UVlight (254 and 366 nm). Silica gel for flash chromatography was MerckKieselgel 60 (230-400 mesh, ASTM) or Sorbent Technologies 60 Å (40-63μm, technical grade). MAL-dPeg™₄-t-boc-hydrazide was obtained fromQuanta Biodesign, Ltd. (Powell, Ohio) and used as received. Cy3 NHS dyeand PD-10 Desalting columns were received from GE Healthcare(Piscataway, N.J.). Spectra/Por® Biotech Cellulose Ester (CE) DialysisMembranes (1,000 MWCO) obtained from Spectrum Laboratories, Inc. (RanchoDominguez, Calif.). SnakeSkin® Pleated Dialysis Tubing (10,000 MWCO) wasobtained from Pierce Biotechnology, Inc. (Rockford, Ill.).

Instrumentation:

Samples were centrifuged at 600 rpm on a Model CS InternationalCentrifuge from International Equipment Company (Boston, Mass.).Reverse-phase high performance liquid chromatography (RP-HPLC) wascarried out with a Varian Prostar HPLC. The products were eluted using asolvent gradient (solvent A=0.05% TFA/H₂O; solvent B=0.05% TFA/CH₃CN).Nuclear magnetic resonance was performed on Bruker AC300 and AC400Fourier Transform Spectrometers using deuterated solvents and thesolvent peak as a reference. Gel permeation chromatography was performedin tetrahydrofuran (THF) with the eluent at a flow rate of 1 mL/min on aWaters chromatograph equipped with four 5 mm Waters columns (300 mm×7.7mm) connected in series with increasing pore size (100, 1000, 100,000and 1,000,000 Å respectively). A Waters 2487 Dual X Absorbance Detectorand a 2414 Refractive Index Detector were employed. Dynamic lightscattering was performed on a Malvern Zetasizer Nanoseries instrumentwith a CGS-3 compact goniometer system.

17. Synthesis of Compound 1

To a solution of dimethoxyethane (40 mL) was added MeNO₂ (11.37 mL, 200mmol) followed by Triton B (2 mL). The mixture was heated to 67° C. andthen tert-butyl acrylate (91.83 mL, 620 mmol) was added to maintain thetemperature at 75° C. When the temperature started to decrease,additional Triton B (1 mL) was added. After the addition was completed,the solution was heated to maintain at 75° C. for 2 hours. The solventwas removed in vacuo and the residue was dissolved in CHCl₃ and theresulting organic solution was washed with 10% HCl, brine, and driedover anhydrous Na₂SO₄. Removal of the solvent in vacuo gave a crudesolid that was further purified by recrystallization from EtOH to obtaina colorless crystal (95% yield). ¹H NMR (CDCl₃): δ1.44 (s, CH₃, 27H),2.21 (m, CH₂, 12H). ¹³C NMR (CDCl₃): 27.93 (CH₃), 29.68 (CH₂CO), 30.22(CCH₂), 81.02 (CCH₃), 92.09 (CNH₂), 170.97 (CO₂).

18. Synthesis of Compound 2

A solution of compound 1 (6.0 g, 0.0135 mol) in a mixture of ethanol(140 mL) and dichloromethane (20 mL) was added to a Parr hydrogenationbottle. Then, 4 grams of Raney-nickel was added. The mixture washydrogenated at 50 psi and room temperature. The reaction was monitoredby thin-layer chromatography (TLC) until the starting materialdisappeared. The catalyst was carefully filtered through Celite, and thesolvent was removed in vacuo yielding a crude solid. The residue wasdissolved in dichloromethane and washed with saturated NaHCO₃ and water,and then dried over anhydrous Na₂SO₄. Removal of dichloromethane gave awhite solid (93%). ¹H NMR (CDCl₃): δ1.44 (s, CH₃, 27H), 1.95 (t, CH₂,6H), 2.43 (t, CH₂, 6H); ¹³C NMR (CDCl₃): 27.98 (CH₃), 29.46 (CH₂CO),31.47 (CCH₂), 56.99 (CNH₂), 80.96 (CCH₃), 172.30 (CO₂).

19. Synthesis of Compound 4

To a solution of compound 3 (0.65 g, 2.35 mmol) in 50 mL dry THF thefollowing reagents were added 1-hydrobenzotriazole (HOBt) (0.96 g, 7.10mmol), DCC (1.46 g, 7.10 mmol) and then 2 (3.54 g, 8.5 mmol). Thesolution was stirred at room temperature and the reaction was monitoredby TLC. After 40 hrs, the white precipitate was filtered and thesolution was concentrated to yield a crude residue. The product waspurified by column chromatography (silica gel, hexane:ethyl acetate=3:2)yielding a white solid (85%). ¹H NMR (CDCl₃): δ1.44 (m, CH₃, 81H), 1.95(m, CH₂, 18H), 2.21 (m, CH₂, 30H), 6.20 (s, NH, 3H); ¹³C NMR (CDCl₃):28.04, 29.74, 29.85, 31.28, 57.56, 80.69, 92.47, 170.46, 172.76.

20. Synthesis of Compound 5

A solution of compound 4 (1.47 g, 1 mmol) in 15 mL of formic acid wasstirred at room temperature overnight. After the solution wasconcentrated, toluene was added and the solution was evaporated toremove any residue of formic acid to give a white solid (100%). ¹H NMR(DMSO): δ1.81 (m, CH₂, 18H), 2.11 (m, CH₂, 30H), 7.29 (s, NH, 3H), 12.10(br, COOH); ¹³C NMR (DMSO): 28.03, 29.03, 30.08, 56.41, 93.31, 170.43,174.42.

21. Synthesis of Compound 6

To a solution of compound 5 (2.12 g, 0.0022 mol) in DMF (30 mL), HOBt(2.68 g, 0.0198 mol) and DCC (4.09 g, 0.0198 mol) were added. Themixture was chilled to 0° C. with ice-water bath. Then, a solution ofN-Boc-ethylenediamine (3.49 g, 0.0218 mol) in DMF (5 mL) was addeddropwise at 0° C. The reaction mixture was stirred at room temperaturefor 48 hrs. The solution was then filtered and 200 mL of dichloromethanewas added, and washed with 1N HCl, saturated NaHCO₃, and water. Theorganic phase was dried over anhydrous Na₂SO4 and evaporated to yield acrude residue. The product was purified by column chromatography (elutedfirst with 2% methanol in dichloromethane, then with 6% methanol indichloromethane, followed by 10% methanol in dichloromethane) to obtaina white solid (51%). ¹H NMR (CD₃OD): δ 1.44 (m, CH₃, 81H), 1.80-2.10 (m,CH₂, 48H), 3.0-3.2 (m, CH₂, 36H), 6.20 (m, NH, 3H), 6.46 (m, NH, 8H),7.71 (m, NH, 8H); ¹³C NMR (CD₃OD): 28.40, 31.24, 31.44, 31.80, 32.09,40.66, 40.97, 59.14, 80.13, 94.42, 158.48, 173.48, 175.91. This whitesolid was then dissolved in 40 mL of 1, 4-dioxane. At 0° C., 40 mL of 4M HCl in dioxane was added to the solution under Ar atmosphere andstirred at room temperature for 1 hr. Removal of the solvent gave awhite solid as the deprotected HCl salt (100%). ¹H NMR (D₂O): δ1.70-2.15 (m, CH₂, 48H), 3.30 (m, CH₂, 18H), 3.36 (m, CH₂, 18H); ¹³C NMR(D₂O): 27.61, 27.98, 28.86, 35.11, 37.41, 56.29, 92.01, 171.84, 174.98.1.53 g (0.92 mmol) of the resulting HCl salt was dissolved in 80 mL ofmethanol. At 0° C., 3.5 mL of Et₃N was added to the solution, followedby the addition of N,N′-diBoc-N″-triflylguanidine (4.2 g, 10.73 mmol).The solution was stirred at room temperature for 24 hr. After removal ofthe solvent, the residue was dissolved in dichloromethane and washedwith water, 1N HCl, saturated NaHCO₃, and water. The organic layer wasdried over anhydrous Na₂SO₄ and removed in vacuo. The residue productwas purified by column chromatography (eluted with 2% methanol indichloromethane, then 10% methanol in dichloromethane) to give a whitesolid (90%) as compound 6. ¹H NMR (CD₃OD): δ 1.45 (m, CH₃, 81H), 1.51(m, CH₃, 81H), 1.90-2.25 (m, CH₂, 48H), 3.30-3.52 (m, CH₂, 36H); ¹³C NMR(CD₃OD): 28.37, 28.67, 31.32, 31.67, 32.06, 39.74, 41.24, 59.02, 80.23,84.35, 94.31, 153.91, 157.737, 164.38, 173.33, 175.87.

22. Synthesis of Compound 7

To a solution of compound 5 (1.2, 0.001245 mol), HOBt (1.514 g, 0.0112mol) and DCC (2.311 g, 0.0112 mol) were added in 20 mL of DMF. Then,N-Boc-1,6-diaminohexane (2.66 g, 0.0123 mol) was dissolved in 5 mL ofDMF dropwise at 0° C. The solution was then stirred at room temperaturefor 48 hrs. The solution was then filtered and 200 mL of dichloromethanewas added, and washed with 1N HCl, saturated NaHCO₃, and water. Theorganic phase was dried over anhydrous Na₂SO₄ and evaporated to yield acrude residue. The product was purified by column chromatography (elutedfirst with 2% methanol in dichloromethane, then with 5% methanol indichloromethane, followed by 10% methanol in dichloromethane) to obtaina white solid (45%). ¹H NMR (CD₃OD): δ 1.2-1.6 (m, CH₃, CH₂, 153H),1.80-2.10 (m, CH₂, 48H), 3.0-3.2 (m, CH₂, 36H); ¹³C NMR (CD₃OD): 27.54,28.85, 30.37, 30.90, 31.28, 31.60, 32.14, 40.58, 41.24, 59.13, 79.30,94.30, 158.49, 173.50, 175.56. This white solid was then dissolved in 40mL of 1, 4-dioxane. At 0° C., 40 mL of 4 M HCl in dioxane was added tothe solution under Ar atmosphere and stirred at room temperature for 1hr. Removal of the solvent gave a white solid as the deprotected HClsalt (100%). ¹H NMR (D₂O): δ 1.10-1.60 (m, CH₂, 72H), 1.7-2.2 (m, CH₂,48H), 3.30 (m, CH₂, 18H), 3.36 (m, CH₂, 18H). 0.838 g (0.385 mmol) ofthe resulting HCl salt was dissolved in 80 mL of methanol. At 0° C.,1.45 mL of Et₃N was added to the solution, followed by the addition ofN,N′-diBoc-N″-triflylguanidine (1.765 g, 4.51 mmol). The solution wasstirred at room temperature for 24 hr. After removal of the solvent, theresidue was dissolved in dichloromethane and washed with water, 1N HCl,and water. The organic layer was dried over anhydrous Na₂SO₄ and removedin vacuo. The residue product was purified by column chromatography(eluted with 2% methanol in dichloromethane, then 10% methanol indichloromethane) to give a white solid (90%) as compound 7. ¹H NMR(CD₃OD): ¹H NMR of 9 (CD₃OD): δ 1.15-1.55 (m, 234H), 1.70-2.15 (m, CH₂,48H), 3.29-3.30 (m, CH₂, 36H); ¹³C NMR (CD₃OD): 27.70, 27.62, 28.33,28.67, 30.08, 30.33, 31.30, 31.60, 40.48, 40.62, 41.27, 54.5, 59.14,80.25, 84.40, 154.22, 157.49, 164.53, 173.50, 175.53.

23. Synthesis of Compound 8 and 9

Compound 6 (or 7, 0.10 mmol) was dissolved in 40 mL of ethanol andtransferred into a hydrogenation bottle containing 5 g of Raney-Nickelcatalyst. The solution was hydrogenated at room temperature at 65 psiand monitored by TLC. The catalyst was filtered through Celite. Thesolvent was removed in vacuo to give a white solid 8 or 9 (80%). ¹H NMRof 8 (CD₃OD): δ 1.46 (m, CH₃, 81H), 1.51 (m, CH₃, 81H), 1.90-2.25 (m,CH₂, 48 H), 3.30-3.55 (m, CH₂, 36H); ¹³C NMR (CD₃OD): 28.37, 28.67,31.40, 31.76, 39.76, 41.27, 54.0, 58.86, 80.32, 84.37, 153.97, 157.81,164.4, 175.61, 176.02. ¹H NMR of 9 (CD₃OD): δ 1.20-1.70 (m, 234H),1.85-2.40 (m, CH₂, 48H), 3.10-3.50 (m, CH₂, 36H); ¹³C NMR (CD₃OD):27.01, 27.18, 28.27, 28.53, 29.42, 29.71, 30.15, 30.88, 31.19, 40.03,41.23, 54.3, 58.21, 79.93, 83.84, 153.62, 156.65, 163.83, 175.77.

24. Synthesis of Compound FD-1

FITC (0.14 g, 0.36 mmol), dissolved in 1 mL of DMF, was added to asolution of compound 8 (0.23 g, 0.066 mmol) in a mixture of DMF anddichloromethane. The solution was chilled to 0° C., to which Et₃N (0.092mL, 0.66 mmol) was added. The mixture was stirred overnight at roomtemperature. After removal of DMF in vacuo, the residue was dissolved indichloromethane and washed with 1N HCl and water. The dichloromethanelayer was dried over anhydrous Na₂SO₄ and concentrated to obtain ayellow solid. ¹H NMR (CD₃OD): δ 1.46 (m, CH₃, 81H), 1.51 (m, CH₃, 81H),1.90-2.25 (m, CH₂, 48H), 3.30-3.55 (m, CH₂, 36H), 6.52-6.72 (br, 4H),7.15 (br, 1H), 7.5 (br, 2H), 7.72 (br, 1H), 8.4 (br, 1H). The resultingyellow solid (200 mg, 0.052 mmol) was dissolved in 10 mL of 1,4-dioxane. At 0° C., 10 mL of 4 M HCl in dioxane was added to thesolution under Ar protection and stirred at room temperature overnight.After evaporation of the solvent in vacuo, the product was dissolved inwater and the insoluble precipitate was filtered. Removal of wateryielded a crude yellow solid, which was further purified by RP-HPLCusing a solvent gradient (solvent A=0.05% TFA/H₂O; solvent B=0.05%TFA/CH₃CN) to obtain compound 10. ¹H NMR (D₂O): δ 1.85-2.30 (m, CH₂,48H), 3.10-3.30 (m, CH₂, 36H), 6.9 (br, 2H), 7.10-7.2 (m, 3H), 7.4 (s,2H), 7.5 (br, 1H), 8.1 (s, 1H).

25. Synthesis of Compound FD-2

FITC (0.016 g, 0.0376 mmol), dissolved in 1 mL of DMF, was added to asolution of compound 9 (0.050 g, 0.0125 mmol) in a mixture of DMF anddichloromethane (1:1). The solution was chilled to 0° C., to which Et₃N(12 μL) was added. The mixture was stirred overnight at roomtemperature. After removal of DMF in vacuo, the residue was dissolved indichloromethane and washed with 1N HCl and water. The dichloromethanelayer was dried over anhydrous Na₂SO₄ and concentrated to obtain asolid. The product was dissolved in methanol and purified by dialysiswith Spectro® Por Biotech RC membranes (MWCO 3500). After removal of themethanol, a yellow solid was obtained. ¹H NMR (CD₃OD): δ 1.20-1.7 (m,CH₃, CH₂, 234H), 1.89-2.30 (m, CH₂, 48H), 3.10-3.40 (m, CH₂, 36H),6.52-6.72 (br, 4H), 7.15 (br, 1H), 7.5-7.72 (br, 3H), 8.1 (br, 1H). Theresulting yellow solid (200 mg, 0.052 mmol) was dissolved in 10 mL of 1,4-dioxane. At 0° C., 10 mL of 4 M HCl in dioxane was added to thesolution under Ar protection and stirred at room temperature overnight.The precipitate was filtered out and dried in vacuo. The obtained yellowsolid was dissolved in water and lyophilized to yield compound 11. ¹HNMR (D₂O): δ 1.1-1.50, (m, CH₂, 72H), 1.50-2.20 (m, CH₂, 48H), 3.10-3.30(m, CH₂, 36H), 6.5-6.7 (br, 6H), 7.10 (m, 1H), 7.5 (br, 3H).

26. Examples FD-1 and FD-2

As examples of the compounds of the invention, two non-peptidicfluorescently labeled Newkome-type dendrimers, differentiated over avaried alkylspacer with guanidine end moieties, were designed andsynthesized. The assessment of internalization into mammalian cellsusing NIH-3T3 fibroblasts and human microvascular endothelial cells(HMEC) showed that the spacer length at the terminal generation of thedendrimers can affect direction of cargo molecules precisely intospecific subcellular compartments (e.g., nucleus or cytosol). Suchdirection can be particularly advantageous for the controlledintracellular delivery of bioactive cargo molecules into targetedlocations.

The two exemplary FITC-dendrimer conjugates were found to be highlywater soluble and were further investigated for their capability totranslocate through the cell membrane. Internalization of FD-1 and FD-2in mammalian cells was assessed using two different cell lines and apreviously described method [Futaki, S.; Nakase, I.; Suzuki, T.; Youjun,Y.; Sugiura, Y. Biochemistry 2002, 41, 7925.] with NIH-3T3 fibroblastsand HMEC (human microvascular endothelial cells) and a Zeiss LSM 510confocal microscope. FIG. 21 shows the time course of uptake of FD-1 andFD-2 into NIH-3T3 Fibroblasts at 37° C. The fluorescence was clearlyobserved within the cells 2.5 min after the addition of conjugates tothe medium, which is comparable to the uptake rate of Tat-peptide.[Futaki, S.; Nakase, I.; Suzuki, T.; Youjun, Y.; Sugiura, Y.Biochemistry 2002, 41, 7925.; Vives, E.; Brodin, P.; Lebleu, B. J. Biol.Chem. 1997, 272, 16010.] Furthermore, the extent of internalizationincreased in an incubation time-dependent manner, and it was observedthat after just 10 min, the fluorescence intensity of cells treated withFD-2 was near saturation. However, the fluorescence intensity of cellstreated with FD-1 did not approach saturation until the longer timepoints (45 min˜2 hr). Additionally, FD-1 and FD-2 exhibited differentialpatterns of subcellular localization, as FD-1 appeared to concentrate inthe nucleus while FD-2 appeared to concentrate in the cytosol. Withoutwishing to be bound by theory, it is believed that the length of thespacer at the terminal generation of the dendrimer can not only controlthe uptake rate, [Wender, P. A.; Kreider, E.; Pelkey, E. T.; Steinman,L.; Rothbard, J. B.; VanDeusen, C. L. Org. Lett. 2005, 7, 4815.] butalso regulate the subcellular localization of the molecule and itsputative cargo. For instance, the uptake levels of FD-2 appeared to begenerally stronger than those of FD-1 after the same incubation time atthe same concentration. Therefore, the dendrimer with a hexyl spacercrosses the cell membrane faster than the molecule with an ethyl chain.On the other hand, the localization patterns can also be controlled bythe length of the spacer. FD-1 with the short spacer appeared to belocalized everywhere in the cell, but highly concentrated in thenucleus. However, FD-2, with its longer spacer, was observed to residemainly in the cytosol. These translocation features of guanidinlyateddendritic scaffolds as carriers can be important for intracellulardelivery of cargo molecules to specific subcellular compartments (e.g.,cytosol or nucleus). For example, a translocation approach that does notsaturate the nucleus can be highly attractive as it can be both lesscytotoxic and could afford cytosolic-targeted cargos with greateraccuracy in delivery, and therefore higher efficacy. Without wishing tobe bound by theory, it is believed that the differential uptake patternsby FD-1 and FD-2 are due to the presence of a hexyl spacing chain inFD-2, resulting in a greater hydrophobicity of the entire conjugate ascompared with FD-1. Additionally, the uptake of FD-1 and FD-2 conjugatesby HMEC was also conducted. Entry of the two conjugates into HMEC showsa similar internalization pattern to that seen in fibroblasts.

In control experiments, cells treated with free FITC and Boc-protectedguanidinylated FITC-dendrimer showed no or extremely weak fluorescence,respectively. Therefore, the guanidino groups play an important role inthe cell permeability of these molecules, while the length of thespacing chain determines both the differential rate of uptake andsubcellular localization patterns. Although the mechanism of Tattranslocation remains to be understood, it has been demonstrated thatthe rate of uptake is not temperature dependent. [Futaki, S.; Nakase,I.; Suzuki, T.; Youjun, Y.; Sugiura, Y. Biochemistry 2002, 41, 7925.;Vivés, E.; Brodin, P.; Lebleu, B. J. Biol. Chem. 1997, 272, 16010.] Thisindicates that endocytosis does not play a crucial role in thetranslocation process. Evaluation of the effect of temperature on theinternalization of FD-1 and FD-2 indicated that the two conjugates areable to get into cells not only at 37° C., but also at 4° C., even at alower dendrimer concentration (1 μM) (see FIGS. 3 and 4 in contrast tocontrol experiments, as shown in FIG. 24). No significant decrease influorescence intensity of cells treated with FD-1 or FD-2 was observed,indicating that the uptake process does not occur via endocytosis.

27. Synthesis of Dendrimer B11.

A three-neck round bottom flask was flame-dried under argon, to whichnitrotriacid B3 (3.192 g, 0.0115 mmol), 1-hydrobenzotriazole (HOBt)(5.609 g, 0.0415 mol), DCC (8.560 g, 0.0415 mol) and 100 mL THF wereadded sequentially. After 2 hours activation, aminotriester B2 (17.216g, 0.0415 mol) was added. The solution was stirred at room temperaturefor 40 h, and the crude product was purified by flash columnchromatography, eluting first with hexane/ethyl acetate (10:1) and thenhexane/ethyl acetate (3:2) to yield dendrimer B11 (15.91 g, 94.1%). ¹HNMR (400 MHz, CDCl₃): δ=□1.44 (m, CH₃, 81H), 1.95 (m, CH₂, 18H), 2.21(m, CH₂, 30H), 6.20 (s, NH, 3H); ¹³C NMR (400 MHz, CDCl₃): δ=28.04,29.74, 29.85, 31.28, 57.56, 80.69, 92.47, 170.46, 172.76.

28. Synthesis of Dendrimer B12.

A solution of B11 (10.0 g, 0.0 mol) in 150 mL of absolute ethanol in thepresence of 8 grams of Raney-Nickel was hydrogenated at 60 psi ofhydrogen at room temperature for 24 h. The suspension was carefullyfiltered through Celite and removal of the solvent under reducedpressure yielded B12 (9.86 g, 98.5%). ¹H NMR (400 MHz, CD₃OD): δ=□1.44(m, CH₃, 81H), 1.61 (m, CH₂, 6H), 1.95 (m, CH₂, 12H), 2.21 (m, CH₂, 30H); ¹³C NMR (400 MHz, CD₃OD): δ=28.42, 30.24, 30.47, 32.02, 36.24,53.53, 58.37, 81.18, 173.96, 175.39.

29. Synthesis of B25.

To a room temperature stirred solution of 6-bromohexanoic acid (2.0 g,0.0102 mol) in 7 mL of DMF was added NaN₃ (1.30 g, 0.020 mol). Thereaction mixture was heated and stirred at 85° C. for 5 h. After DMF wasremoved, DCM was added to dissolve the residue. The mixture was washedwith 0.1 N HCl and dried over anhydrous NaSO₄. Removal of the solventgave a crude oil that was purified by flash column chromatography,eluting first with DCM and then ethyl acetate/DCM (3:7) to yield B25(1.67 g, 69.07%). ¹H NMR (400 MHz, MeOD): δ=□1.38-1.49 (m, CH₂, 2H),1.54-1.70 (m, CH₂, 4H), 2.32 (t, CH₂, 2H), 3.30 (t, CH₂, 2 H); ¹³C NMR(400 MHz, MeOD): δ=□25.57, 27.32, 29.62, 34.72, 52.27, 177.38.

30. Synthesis of Dendrimer B13.

To a stirred solution of B25 (1.29 g, 8.22 mmol) in anhydrous THF (50mL) were added DCC (1.70 g, 8.22 mmol) and HOBt (1.112 g, 8.22 mmol) atroom temperature. The mixture was stirred for 2 h, then dendrimer B12(9.86 g, 6.85 mmol) was added and the resulting solution was stirred for40 h. After filtration and removal of THF, the product was purified byflash column chromatography, eluting with hexane/ethyl acetate (1:1) toyield B13 (8.50 g, 78.53%). ¹H NMR (400 MHz, CD₃OD): δ=□1.44 (m, CH₃,CH₂, 83H), 1.95 (m, CH₂, 18H), 2.21 (m, CH₂, 32H), 3.30 (m, CH₂, 2 H);¹³C NMR (400 MHz, CD₃OD): δ=26.47, 27.47, 28.43, 29.62, 30.35, 30.61,32.07, 32.23, 37.56, 52.28, 58.63, 58.77, 81.54, 174.21, 175.35, 175.66.

31. Synthesis of Dendrimer B14.

To a 0° C. stirred solution of nona-amine B5 (4.06 g, 2.43 mmol) in amethanol/acetonitrile (25 mL/15 mL) were added triethylamine (6.87 g,68.0 mmol) and ethyl trifluoroacetate (9.32 g, 65.6 mmol) and thereaction mixture was stirred at 0° C. for 1 h and then at roomtemperature overnight. The solvent was removed in vacuo and the residuewas taken up in ethyl acetate, and the resulting organic solution waswashed with 1N HCl and brine and dried over anhydrous NaSO₄. Removal ofthe solvent in vacuo gave a crude solid that was purified by flashchromatography (EtOAc/Methanol gradient) to yield a solid (3.02 g,56.3%). ¹H NMR (400 MHz, CD₃OD): δ=□1.85-2.10 (m, CH₂, 18H), 2.11-2.35(m, CH₂, 30H), 3.24-3.48 (m, CH₂, 36 H); ¹³C NMR (400 MHz, CD₃OD):δ=□31.08, 31.26, 31.75, 32.01, 39.42, 40.42, 58.93, 94.33, 111.74,115.54, 119.33, 123.13, 158.57, 159.06, 159.55, 160.04, 173.57, 176.14.The resulting white solid (1.0 g, 0.453 mmol) was dissolved in ethanol(45 mL) and transferred into a hydrogenation vessel containingRaney-Nickel catalyst (5 g) and the suspension was stirred at 80 psi ofhydrogen at 50° C. for 48 h. After filtration through Celite, thesolvent was removed under reduced pressure to give a B14 as a whitesolid (0.964 g, 97.7%). ¹H NMR (400 MHz, CD₃OD): δ=1.67□ (m, □CH₂, 6H),□□986 (m, □CH₂, 12H), 2.188 (m, CH₂, 30H), 3.30-3.55 (m, CH₂, 36 H); ¹³CNMR (400 MHz, CD₃OD) δ=31.20, 32.11, 36.17, 39.38, 40.52, 54.06, 58.80,111.79, 115.53, 119.36, 123.10, 158.58, 1 59.04, 159.50, 160.11, 175.55,176.24.

32. Synthesis of Dendrimer B15.

To a stirred solution of 6-heptynoic acid (0.3022 g, 2.40 mmol) inanhydrous THF (50 mL) were added DCC (0.4952 g, 2.40 mmol) and HOBt(0.3245 g, 2.40 mmol) at room temperature. The mixture was stirred for 2h, then dendrimer B14 (1.0432 g, 0.48 mmol) was added and the resultingsolution was stirred for 40 h. After filtration and removal of THF, theproduct was purified by flash column chromatography, eluting with ethylacetate/methanol gradient to yield B15 (0.620 g, 56.57%). ¹H NMR (400MHz, CD₃OD): δ=1.53 (m, CH₂, 2H), 1.71 (m, CH₂, 3H), 1.890-2.5 (m, CH₂,50H), 3.30 (m, CH₂, 36 H); ¹³C NMR (400 MHz, CD₃OD): δ□=□18.81, 26.14,29.43, 31.27, 31.80, 37.37, 39.35, 40.43, 58.83, 59.05, 69.95, 83.4,111.74, 115.57, 119.37, 123.13, 158.55, 159.07, 159.53, 159.99, 175.60,176.25.

33. Synthesis of Dendrimer B16.

Azide dendron B13 (100 mg, 0.044 mmol) and alkyne dendron B15 (70 mg,0.044 mmol) were dissolved in THF/H₂O (4:1) and DIPEA (0.017 g, 0.132mmol, 3 equiv) followed by Cu(PPh₃)₃Br (0.0042 g, 0.0044 mmol) wereadded. The reaction mixture was placed in the microwave reactor(Biotage) and irradiated at 120° C. for 20 min. After completion of thereaction, THF was removed and the residue was taken up in DCM. Theorganic layer was washed with water once and dried over anhydrousNa₂SO₄. ¹H NMR of B16 (400 MHz, CD₃OD): δ=□1.43 (m, CH₃, 81H), 1.71 (m,CH₂, 8H), 1.890-2.5 (m, CH₂, 96H), 2.71 (m, CH₂, 2H), 3.30 (m, CH₂,36H), 4.38 (m, CH₂, 2H), 7.75 (s, 1H).

34. Synthesis of B17 and B18.

The “Bow-Tie” B16 was stirred in formic acid overnight at roomtemperature. After the solvent was evaporated under reduced pressure,toluene was added and concentrated in vacuo to remove any residue offormic acid to give a white nonacid (100%). To a solution of the aboveresulting solid in DMF, HOBt and DCC were added and the solution wascooled to 0° C. N-Boc-ethylenediamine or N-Boc-hexyldiamine was addeddropwise and the mixture was stirred for 48 h at room temperature,filtered and concentrated under reduced pressure. The residue wasdissolved in dichloromethane and the resulting organic solution waswashed sequentially with 1N HCl, water and dried over anhydrous Na₂SO₄.The solvent was evaporated under reduced pressure and the crude residuewas purified by flash column chromatography to yield B17 or B18.

35. Synthesis of B19 and B20.

Potassium carbonate was added to B17 or B18 in methanol/water, themixture was stirred at room temperature for 6 h. The crude product waspurified by dialysis against methanol with Spectra® Por Biotechregenerated cellulose membranes (MWCO=3500) for 24 h to give B19 or B20.

36. Synthesis of B21 and B22.

The above B19 or B20 was then dissolved in 1, 4-dioxane and the solutioncooled 0° C., 4 M HCl in dioxane was added and stirred for 1 hr at roomtemperature. Removal of the solvent under reduced pressure gave a whitesolid. The resulting HCl salt was dissolved in methanol and the solutionwas cooled to 0° C. Et₃N was added, followed byN,N′-diBoc-N″-triflylguanidine and the mixture was stirred for 24 h atroom temperature. After the solvent was evaporated under reducedpressure, the residue was dissolved in dichloromethane and the solutionwas washed with 1N HCl water, and dried over anhydrous Na₂SO₄. Afterremoval of the solvent under reduced pressure, the crude product waspurified by dialysis against methanol with Spectra® Por Biotechregenerated cellulose membranes (MWCO=3500) for 24 h to give B21 or B22.

37. Synthesis of B23 and B24.

The resulting solid B21 or B22 was dissolved in 1,4-dioxane and thesolution cooled to 0° C., 4 M HCl in dioxane was added and the solutionstirred overnight at room temperature. The precipitate was filtered offand dried to give a crude product. The solid was re-dissolved in waterand insoluble precipitate was filtered off and the filtrate was dialyzedagainst water with Spectra® Por Biotech cellulose ester membranes(MWCO=1000) for 48 hrs and lyophilized to yield a water-soluble B23 orB24.

Synthesis of Copolymer poly(vl-avl-opd) (AbD)

To a 25 mL 3-necked round bottom flask, equipped with stir bar, gasinlet and 2 rubber septa, 2-oxepane-1,5-dione (0.7000 g, 5.46 mmol) wasadded. The round bottom flask was purged with argon. After purging for30 min, dry toluene (4 mL) was added. The mixture stirred in an oil bathat 80° C. to dissolve the monomer. Upon dissolving, Sn(Oct)₂ (0.011 g,2.73×10⁻² mmol) in 0.5 mL dry toluene, absolute ethanol (0.020 g,4.4×10¹ mmol), α-allyl-δ-valerolactone (1.15 g, 8.2 mmol) andδ-valerolactone (1.37 g, 13.7 mmol) were then added to the reactor andthe mixture was heated for 48 h at 105° C. Residual monomer and catalystwere removed by dialyzing with Spectra/Por® dialysis membrane(MWCO=1000) against CH₂Cl₂ to give a golden brown polymer. Yield: 1.7 g.M_(w)=3287 Da, PDI=1.17; ¹H NMR (300 MHz, CDCl₃/TMS, ppm) δ: 5.72 (m,H₂C═CH—), 5.06 (m, H₂C═CH—), 4.34 (m, —CH₂CH₂C(O)CH₂CH₂O—), 4.08 (m,—CH₂O—), 3.67 (m, —OCH₂CH₃), 2.78 (m, opd, —OC(O)CH₂CH₂C(O)CH₂—), 2.58(m, opd, —OC(O)CH₂CH₂C(O)CH₂—), 2.34 (m, vl, —CH₂CH₂C(O)O—, avl,H₂C═CHCH₂CH—, H₂C═CHCH₂CH—), 1.66 (m, avl & vl, —CHCH₂CH₂—), 1.25 (t,—CH₂CH₃); ¹³C NMR (400 MHz, CDCl₃, ppm) δ: 204.9, 175.2, 173.7, 173.2,135.0, 117.0, 63.9, 44.8, 36.4, 33.6, 28.0, 26.3, 21.3. (10.39% avl,7.97% evl, 6.42% opd and 75.21% vl).

38. Synthesis of poly(vl-evl-avl-opd) (ABbD).

In a 200 mL round bottom flask, equipped with stir bar, poly(vl-avl-opd)(1.7 g, 1.56 mmol) was dissolved in 30 mL CH₂Cl₂. To this solution,3-chloroperoxybenzoic acid (0.2210 g, 1.28 mmol) was added slowly. Themixture was stirred for 72 h at room temperature and then concentratedvia rotary evaporator. The crude product was dissolved in a minimalamount of THF (5 mL) and poured into a round-bottomed flask containing 1L diethyl ether. The solution was kept overnight at 0° C. and a whitesolid was obtained. The solution was decanted off and the solid wasdried in vacuo to obtain poly(avl-evl-vl-opd). Yield: 1.2 g (71%). ¹HNMR (300 MHz, CDCl₃/TMS, ppm) δ: 5.72 (m, H₂C═CH—), 5.06 (m, H₂C═CH—),4.34 (m, —CH₂CH₂C(O)CH₂CH₂O—), 4.08 (m, —CH₂O—), 3.67 (m, —OCH₂CH₃),2.96 (m, epoxide proton), 2.78 (m, evl epoxide proton, opd,—OC(O)CH₂CH₂C(O)CH₂—), 2.58 (m, opd, —OC(O)CH₂CH₂C(O)CH₂—), 2.47(epoxide proton), 2.34 (m, vl, —CH₂CH₂C(O)O—, avl, H₂C═CHCH₂CH—,H₂C═CHCH₂CH—), 1.66 (m, avl & vl, —CHCH₂CH₂—), 1.25 (t, —CH₂CH₃).

39. Nanoparticle Formation from poly(vl-evl-avl-opd).

In a 250 mL three-necked round bottom flask equipped with stir bar,condenser and septa, a solution of 2,2′-(ethylenedioxy)diethylamine(26.4 μL, 0.18 mmol) in 55.6 mL CH₂Cl₂ was heated at 44° C. A solutionof poly(avl-evl-vl-opd) (0.2500 g, M_(w)=3287 Da, PDI=1.17) dissolved inCH₂Cl₂ (0.36 mL) was added dropwise via a peristaltic pump at 13 mL/minwith vigorous stirring. The reaction mixture was heated for 12 h.Residual diamine was removed by dialyzing with SnakeSkin® PleatedDialysis Tubing (MWCO=10,000) against dichloromethane. ¹H NMR (300 MHz,CDCl₃/TMS, ppm) δ: The significant change is the disappearance of theepoxide protons at 2.96, 2.75 and 2.47 ppm and the appearance of signalsat 3.5 and 2.89 ppm corresponding to the protons neighboring thesecondary amine of the PEG linker after cross-linking. All other aspectsof the spectrum are similar. To demonstrate the reactivity of the allygroups to thiols, in a model reaction we added benzyl mercaptan to theallyl groups. We found a high reactivity using no other reactant. Wealso added the molecular transporter in the same fashion.

40. Attachment of Benzyl Mercaptan to poly(vl-evl-avl-opd) Nanoparticles(General Procedure to Attach Thiol Functionalized Compounds Including“Molecular Transporter” and Peptides)

In a vial equipped with a stir bar, poly(avl-evl-vl-opd) nanoparticles(0.030 g, 0.0268 mmol) and benzyl mercaptan (9.48 mg, 0.0764 mmol) weredissolved in 0.6 mL toluene. The reaction mixture was heated for 72 h at30° C. The remaining toluene was removed in vacuo and residual benzylmercaptan was removed by dialyzing with SnakeSkin® Pleated DialysisTubing (MWCO=10,000) against dichloromethane. ¹H NMR (300 MHz,CDCl₃/TMS, ppm) δ: The significant change is the disappearance of theallyl protons at 5.72 and 5.06 ppm and the appearance of signals at 3.52ppm and 7.30 ppm corresponding to the methylene and benzene protonsrespectively of the attached benzyl mercaptan. All other aspects of thespectrum are similar.

41. Attachment of N-boc-ethylenediamine to Succinimidyl2-vinylsulfonylethyl Carbonate (SVEC).

To a solution of SVEC (1.03 g, 3.72 mmol) in acetonitrile (50 mL),N-boc-ethylenediamine (0.77 mL, 4.86 mmol) and water (50 mL) were added.Sodium bicarbonate (0.4066 g, 4.84 mmol) was added and the reactionstirred for 4 h at room temperature. The acetonitrile was removed invacuo and the remaining aqueous phase was diluted with brine (45 mL).The aqueous phase was extracted three times with dichloromethane (90mL). The organic phases were combined, washed with brine, dried withMgSO₄, and concentrated in vacuo. The crude product was purified byflash chromatography (eluent:ethyl acetate) to give a white solid in 90%yield. ¹H NMR (300 MHz, CDCl₃/TMS, ppm) δ: 6.6 (m, H₂C═CH—), 6.4 & 6.17(m, H₂C═CH—), 4.43 (t, —CH₂CH₂OC(O)—), 3.3 (t, —CH₂CH₂OC(O)—), 3.24 (m,—NHCH₂CH₂NHC(O)—), 1.41 (s, —NHC(O)OC(CH₃)₃).

42. Attachment of Sulfonyl Linker to Nanoparticles frompoly(vl-evl-opd).

In a 100 mL round bottom flask, equipped with stir bar, poly(vl-evl-opd)(ABD) nanoparticles (84.6 mg, 2.45×10⁻⁷ mol) were dissolved in 12.5 mLCH₂Cl₂. To this solution, sulfonyl linker (69 μL of 0.85 M linker inmethanol, 5.89×10⁻⁵ mol), NaCNBH₃ (0.0111 g in 0.1 mL methanol,1.77×10⁻⁴ mol) and methanol (12.4 mL) were added. The pH was adjusted to6.5 using 0.1 M hydrochloric acid aqueous solution and 0.1 M sodiumhydroxide aqueous solution. The reaction mixture stirred for 25 h atroom temperature and was purified by dialyzing with SnakeSkin® PleatedDialysis Tubing (MWCO=10,000) against 1:1 dichloromethane/methanol. ¹HNMR (300 MHz, CDCl₃/TMS, ppm) δ: The significant change is theappearance of the following peaks: 6.8 (m, CH₂═CH—), 6.5 & 6.3 (m,CH₂═CH—), 4.5 (m, CH₂═CHSO₂CH₂CH₂—), 3.3 (m, —NHCH₂CH₂NH—), 3.1 (m,CH₂═CHSO₂CH₂CH₂—). All other aspects of the spectrum are similar.

43. General Procedure for Attachment of Peptide-Alexa Fluor® 750 toLinker Conjugated Nanoparticles.

In a small vial, equipped with stir bar, peptide (33 μL of 0.013 mg/mLpeptide in phosphate buffer-pH 7.2) and Alexa Fluor® 750 (26.5 μL of 20mg/mL Alexa Fluor® in dimethylformamide were added. The reaction stirredfor 24 h in an aluminum foiled. In a small vial, poly(vl-evl-opd) (ABD)nanoparticles (29.9 mg) were dissolved in 800 μL phosphate buffer(pH=7.2) and 700 μL dimethylformamide. To the peptide-Alexa Fluor®solution, 251 μL of dissolved nanoparticles was added. After stirringfor 45 min at room temperature, additional peptide (2 mg, 1.84×10⁻⁶ mol)was added. The reaction mixture was purified using concentrator tubeswith a molecular weight cut-off of 10,000 Da.

44. Attachment of Alexa Fluor® 750 to poly(vl-evl-opd) Nanoparticles.

In a 25 mL round bottom flask, poly(vl-evl-opd) nanoparticles (63.55 mg,1.92×10⁻⁷ mol) was dissolved in 6.4 mL tetrahydrofuran. The round bottomflask was sealed with a rubber septum and purged with argon. To thepurged solution, Alexa Fluor® 750 (5 mg in 0.5 mL anhydrousdimethylformamide was added. The reaction mixture stirred for 24 h atroom temperature. After 24 h, N-acetoxy succinimide (50 mg, 0.3 mmol)was added to quench the remaining unreacted amines. ¹H NMR (300 MHz,CDCl₃/TMS, ppm) δ: The significant change is the appearance of thefollowing peaks: 7.12, 5.6, 5.5, 5.1, 3.81, 1.90 ppm. The structure ofAlexa Fluor® 750 is not publicly known. All other aspects of thespectrum are similar.

45. General Reductive Amination for the Attachment of Peptides to AlexaFluor® Conjugated Nanoparticles.

In a small vial, equipped with stir bar, peptide (2.6 mg, 2.4×10⁻⁶ mol)was dissolved in 2 mL tetrahydrofuran. To this solution, dye conjugatednanoparticles (0.0923 g, 2.8×10⁻⁸ mol, in 0.5 mL tetrahydrofuran) andNaCNBH₃ (2.23 μL of 1.0 M NaCNBH₃ in tetrahydrofuran) were added. Thereaction mixture stirred for 12 h at room temperature. The reactionmixture was purified by dialyzing with SnakeSkin® Pleated DialysisTubing (MWCO=10,000) against tetrahydrofuran. ¹H NMR (300 MHz,CDCl₃/TMS, ppm) δ: The significant change is the appearance of thefollowing peaks: 5.2, 5, 4.8, 2.6, 2.45, 2.0, 1.22 and 0.89 ppm.

46. Attachment of N-(boc)-2,2(ethylenedioxy)diethylamine.

A 50 mL 3-neck round bottom flask was flame-dried under argon. Thedeprotected nanoparticles (27.6 mg, 0.79 μmol) were dissolved in DriSolvDMF and transferred to the sealed flask, which was then cooled to 0° C.via an ice bath. N-methylmorpholine (6.37 mg, 0.063 mmol) followed byisobutyl chloroformate (9.46 mg, 0.0693 mmol) was added to the cooledsolution and allowed to activate for 1.5 h. Next,N-(boc)-2,2(ethylenedioxy)diethylamine (15.6 mg, 0.063 mmol) was added,the ice bath was removed and the reaction was allowed to stir overnight.The reaction was concentrated in vacuo, the residue was dissolved inMeOH, transferred to SnakeSkin® Pleated Dialysis Tubing (MWCO=10 000),and was dialysed against MeOH.

47. Deprotection of Nanoparticles ContainingN-(Boc)-2,2(ethlenedioxy)diethylamine.

The nanoparticles were dissolved in 2 M HCl/Dioxane (15 mL). Thereaction was allowed to stir overnight. The reaction was dissolved inMeOH/H₂O and transferred to SnakeSkin® Pleated Dialysis Tubing (MWCO=10000), and was dialysed against MeOH/H₂O.

48. Attachment of Alexa Fluor 750®.

PBS Buffer (pH 7.3) was purged with argon for 1 h. The Alexa Fluor® 750(3 mg, 2.3 μmol) in 0.3 mL DMF was added to a solution of deprotectednanoparticles (15.8 mg) in PBS Buffer (1.2 mL) and was allowed to stirfor 24 h. The reaction was diluted with H₂O, was transferred toSnakeSkin® Pleated Dialysis Tubing (MWCO=10 000), and was dialyzedagainst H₂O.

49. Attachment of SVEC.

The nanoparticles were dissolved in 4 mL of H₂O followed by the additionof sodium bicarbonate (2.7 mg, 0.0318 mmol). Next, the SVEC was added in1 mL of ACN followed by an additional 3 mL of ACN. The reaction wasallowed to proceed for 2 h at which time acetoxysuccinimide (127 mg,0.79 mmol) was added in order to quench any remaining amines. Thisreaction was allowed to proceed for 2 h. The reaction was diluted withH₂O and was transferred to SnakeSkin® Pleated Dialysis Tubing (MWCO=10000), and was dialyzed against H₂O (pH 4.5).

50. Targeting Peptide Attachment.

The modified nanoparticles (2 mg) were dissolved in 0.2 mL of PBS Buffer(pH 7.3) and to that a solution of GCGGGNHVGGSSV (11.4 mg, 0.0105 mmol)in 0.4 mL of PBS Buffer (pH 7.3) was added. This reaction was allowed toproceed for 24 h. The reaction was diluted with H₂O and was transferredto SnakeSkin® Pleated Dialysis Tubing (MWCO=10 000), and was dialyzedagainst H₂O.

51. Control Peptide Attachment.

The modified nanoparticles (2 mg) were dissolved in 0.2 mL of PBS Buffer(pH 7.3) and to that a solution of GCGGGSGVSGHNG (11.0 mg, 0.0105 mmol)in 0.4 mL of PBS Buffer (pH 7.3) was added. This reaction was allowed toproceed for 24 h. The reaction was diluted with H₂O and was transferredto SnakeSkin® Pleated Dialysis Tubing (MWCO=10 000), and was dialyzedagainst H₂O.

52. Attachment of N-(Boc)-2,2(ethylenedioxy)diethylamine.

A 50 mL 3-neck round bottom flask was flame-dried under argon. Thedeprotected nanoparticles (27.6 mg, 0.79 μmol) were dissolved in DriSolvDMF and transferred to the sealed flask, which was then cooled to 0° C.via an ice bath. N-methylmorpholine (6.37 mg, 0.063 mmol) followed byisobutyl chloroformate (9.46 mg, 0.0693 mmol) was added to the cooledsolution and allowed to activate for 1.5 h. Next,N-(boc)-2,2(ethylenedioxy)diethylamine (15.6 mg, 0.063 mmol) was added,the ice bath was removed and the reaction was allowed to stir overnight.The reaction was concentrated in vacuo, the residue was dissolved inMeOH, transferred to SnakeSkin® Pleated Dialysis Tubing (MWCO=10 000),and was dialysed against MeOH.

53. Deprotection of Nanoparticles ContainingN-(Boc)-2,2(ethlenedioxy)diethylamine.

The nanoparticles were dissolved in 2 M HCl/Dioxane (15 mL). Thereaction was allowed to stir overnight. The reaction was dissolved inMeOH/H2O, was transferred to SnakeSkin® Pleated Dialysis Tubing (MWCO=10000), and was dialysed against MeOH/H2O.

54. Attachment of 1,4,7,10-Tetraazacyclododecane-1,4,7-tris(T-butylacetate)-10-succinimidyl Acetate (DOTA).

The nanoparticles were dissolved in DMF followed by the addition oftriethylamine (TEA). To this solution, DOTA was added and the reactionwas allowed to stir overnight. The reaction was concentrated in vacuo,the residue was dissolved in MeOH/H₂O, transferred to SnakeSkin® PleatedDialysis Tubing (MWCO=10 000), and dialyzed against MeOH/H₂O.

55. Deprotection of Nanoparticles Containing T-Butyl Protected DOTA.

The nanoparticles were dissolved in 2 M HCl/Dioxane (15 mL). Thereaction was allowed to stir overnight. The reaction was dissolved inMeOH/H₂O, transferred to SnakeSkin® Pleated Dialysis Tubing (MWCO=10000), and dialyzed against MeOH/H₂O.

56. Attachment of SVEC.

The nanoparticles were dissolved in H₂O followed by the addition ofsodium bicarbonate. Next, the SVEC was added in ACN followed by anadditional ACN. The reaction was allowed to proceed for 2 h at whichtime acetoxysuccinimide was added in order to quench any remainingamines. This reaction was allowed to proceed for 2 h. The reaction wasdiluted with H₂O, was transferred to SnakeSkin® Pleated Dialysis Tubing(MWCO=10 000), and was dialyzed against H₂O (pH 4.5).

57. Modification of Alexa Fluor 750®.

Alexa Fluor 750® (1.43 mg, 1.1 μmol) was dissolved in 143 μL DMSO andadded to cysteamine (0.077 mg, 1.0 μmol) in 30.9 μL of PBS Buffer (pH7.5). The reaction was allowed to proceed overnight.

58. Simultaneous Attachment of Modified Alexa Fluor 750® and Peptide.

The modified nanoparticles were dissolved in PBS Buffer (pH 7.3), whichhad been purged with argon for 20 min. Next, the modified Alexa Fluor750® and one equivalent of GCGGGNHVGGSSV was added and allowed to reactfor 2 h. An additional 4 equivalents of peptide was then added and thereaction stirred overnight. The solution was diluted in H₂O, transferredto SnakeSkin® Pleated Dialysis Tubing (MWCO=10 000), and dialyzedagainst H₂O.

59. Synthesis of Linear RGD.

A typical Fmoc solid phase peptide synthesis was performed to synthesizethe linear peptide. A cysteine preloaded 2-chlorotrityl resin wasemployed. HOBt: HBTU: DIPEA (1:1:2) in DMF was used as the couplingreagent and amino acids were double coupled. A 20% piperdine (v/v) inDMF employed to deprotect the Fmoc. An amino-hexyl spacer was coupled tothe cystine on the resin, followed by glutamic acid, aspartic acid,glycine, arginine, phenylalanine, and finally lysine.

60. Cyclization of RGD.

The peptide was cyclized by utilizing an ODmab group, which allows forthe selective deprotection carboxylic acid side chain of the glutamicacid, which can then be coupled to the N-terminus. The ODmab wasdeprotected using 2% v/v hydrazine-H₂O/DMF added to the resin andallowed to react for 7 min. Next it was washed with 20 mL of DMFfollowed by 10 mL of a 5% v/v DIPEA/DMF solution which was allowed toshake for 10 min. Carboxy activation was achieved through the use of DCC(44.6 mg, mmol) and HOBt (29.2 mg, mmol) was added to 10 mL of DMF andthen added to the resin and allowed to shake for 18 h.

Reagent R was used to deprotect all side groups and cleave the cyclicpeptide from the resin. Reagent R was prepared by combining 5.4 mL TFA,0.3 mL thioanisole, 0.18 mL anisole, and 0.12 mL ethanedithiol. This wasallowed to react for 3 hours at which time the resin was filtered off.The supernatant was cooled to 0° C. and the peptide was precipitatedusing cold diethyl ether. It was collected through centrifugation andthen washed three times using diethyl ether. The pellet was dissolved in0.6 mL H₂O and 0.4 mL ACN with 0.3% TFA and purified using HPLC.

61. Synthesis of N-Boc-N-Tfa-ethylenediamine.

To a solution of N-boc-ethylenediamine (5.0 g, 31.2 mmol) in 20 mL THF,ethyl trifluoroacetate (3.72 mL, 31.2 mmol) was added dropwise and thereaction stirred overnight. The reaction solution was concentrated toyield a white crystalline product (8.0 g, 100%). ¹H NMR (400 MHz, CDCl₃)δ 1.44 (s, 9H, CH₃), 3.37 (dd, 2H, J=5.4 Hz, J=10.2 Hz, CH₂), 3.46 (dd,2H, J=5.1 Hz, J=10.4 Hz, CH₂), 5.01 (s, 1H, NH), 7.85 (s, 1H, NH); ¹³CNMR (400 MHz, CDCl₃) δ 28.2, 39.1, 42.2, 80.6, 140.6, 151.2, 157.7.

62. Boc Deprotection of N-Boc-N-Tfa-ethylenediamine.

N-Boc-N-Tfa-ethylenediamine (8.0 g, 31.5 mmol) was dissolved in 50 mLformic acid and stirred for 14 h at room temperature. After the solventwas evaporated under reduced pressure, toluene was added andconcentrated to remove any residual formic acid, yielding an orange oil(4.90 g, 99.7%). ¹H NMR (400 MHz, MeOD) δ 2.31 (s, 2H, NH₂), 3.15 (t,2H, J=6.1 Hz, CH₂), 3.61 (t, 2H, J=6.1 Hz, CH₂), 8.35 (s, 1H, NH); ¹³CNMR (400 MHz, MeOD) δ 38.5, 39.7, 113.1, 115.9, 118.8, 121.6, 159.7,160.1.

63. Attachment of N-Tfa-ethylenediamine.

The deprotected nanoparticles (162.3 mg, 4.58 μmol) in DriSolv DMF (10.0mL) were stirred under argon at 0° C. with N-methylmorpholine (47.8 mg,472.6 μmol) followed by dropwise addition of isobutyl chloroformate(71.0 mg, 519.8 μmol) in DriSolv DMF (0.75 mL). After 1.5 h, a solutionof N-Tfa-ethylenediamine (73.8 mg, 472.6 μmol) in DriSolv DMF (2.5 mL)was added dropwise. The reaction was allowed to warm to room temperatureand stirred overnight. After removal of DMF in vacuo, the product wasdissolved in methanol and dialyzed against methanol with SnakeSkin®Pleated Dialysis Tubing (MWCO=10,000). ¹H NMR (400 MHz, MeOD) δ7.61-6.45 (br m, aromatic from crosslinker), 3.15-3.00 (br m,N-Tfa-ethylenediamine), 3.00-2.69 (br m, backbone andN-Tfa-ethylenediamine), 2.69-1.34 (br m, backbone).

64. Deprotection OF MAL-dPeg™₄-T-boc-hydrazide.

In a 100 mL round bottomed flask, MAL-dPeg™₄-t-boc-hydrazide (127.1 mg,239.5 μmol) was dissolved in 80.0 mL of formic acid and stirred overnight at room temperature. After the solvent was evaporated underreduced pressure, toluene was added and concentrated to remove anyresidual formic acid to give MAL-dPeg™₄ hydrazide (103.1 mg, 100%).

65. Attachment of Mal-dPeg™₄-Hydrazide.

The deprotected nanoparticles (141.1 mg, 3.13 μmol) in DriSolv DMF (10.0mL) were stirred under argon at 0° C. with N-methylmorpholine (17.1 mg,169.1 μmol) followed by dropwise addition of isobutyl chloroformate(25.4 mg, 86.0 μmol) in DriSolv DMF (0.7 mL). After 1.5 h, a solution ofMAL-dPeg™₄-hydrazide (103.1 mg, 239.5 μmol) was added dropwise. Thereaction was allowed to warm to room temperature and stirred overnight.After removal of DMF in vacuo, the product was dissolved in methanol anddialyzed against methanol with SnakeSkin® Pleated Dialysis Tubing(MWCO=10,000). ¹H NMR (400 MHz, MeOD) δ 7.67-6.47 (br m, aromatic fromcrosslinker and maleimide linker), 3.89-3.48 (br t, maleimide linker),3.21-3.02 (br m, N-Tfa-ethylenediamine), 3.02-2.69 (br m, backbone andN-Tfa-ethylenediamine), 2.69-1.01 (br m, backbone).

66. Hydrogenation of G1.

A solution of G1 (8.36 g, 5.69 mmol) in ethanol (214 mL) in a Parrhydrogenation bottle with Raney-Nickel (3.49 g) was shaken at 65 psi for3 days at room temperature. Another 1 g of Raney-Nickel was added to thereaction and it was again shaken at 65 psi for 3 days at roomtemperature. The reaction was filtered through Celite, and the removalof the solvent under reduced pressure gave the crude product. Theresidue was dissolved in ethyl acetate and subsequently washed withsaturated sodium bicarbonate solution (2×, 100 mL) and brine (2×, 100mL) then the organic layer was dried over anhydrous sodium sulfate. Thesolution was filtered and concentrated under reduced pressure to yieldamine G1 (8.19 g, 93.7%).

67. PDPOH Attachment to G1.

PDPOH (91.46 mg, 4.25 mmol) in dry THF (100 mL) was stirred under argonat room temperature with 1-hydrobenzotriazole (HOBt) (68.90 mg, 5.10mmol) and DCC (1.05 g, 5.10 mmol). After 1 h, amine G1 (7.34 g, 5.10mmol) was added to the solution and the reaction proceeded for 48 h,after which, it was filtered and concentrated under reduced pressure.The crude material was purified via flash column chromatography elutingwith 10:1 hexanes:ethyl acetate increasing to 100% ethyl acetate to givewhite SS-G1 (4.67 g, 67.1%).

68. SSG1 Deprotection Via Formic Acid.

SSG1 (4.67 g, 2.85 mmol) was dissolved with stirring in formic acid (100mL) and the reaction proceeded at room temperature overnight. Uponcompletion, the formic acid was removed azeotropically with tolueneunder reduced pressure to yield the product (3.29 g, 100.0%).

69. N-Boc-1,6-diaminohexane Attachment to SSG1.

SSG1OH (3.29 g, 2.91 mmol) in anhydrous THF (100 mL) was stirred underargon at room temperature with HOBt (4.23 g, 31.25 mmol) and DCC (6.45g, 31.25 mmol). After one hour, N-boc-1,6-diaminohexane (6.76 g, 31.25mmol) was added to the solution and the reaction proceeded for 48 h atroom temperature. Upon completion, the reaction solution was filtered toremove the DCC salt and the filtrate concentrated and purified via flashcolumn chromatography eluting with 1% methanol in dichloromethane andgradually increasing to 10% methanol in dichloromethane to yield a whitesolid (4.42 g, 52.0%).

The resulting solid was dissolved in 1,4-dioxane (20 mL), the solutionwas cooled to 0° C., and 4 M HCl in 1,4-dioxane (20 mL) was added andthe reaction stirred for 24 h at room temperature. Removal of thesolvent under pressure gave a white solid (3.55 g, 100.0%).

70. Attachment of Goodman's Reagent to SSG1LL.

The resulting SSG1LL HCl salt (3.55 g, 1.51 mol) was dissolved inmethanol (50 mL), and the solution was cooled to 0° C. Triethylamine(TEA) (3.41 mL, 24.56 mmol) was added followed byN,N′-diboc-N″-triflylguanidine (6.94 g, 17.74 mmol) and the reaction wasstirred 24 h at room temperature. After removal of the solvent underreduced pressure, the crude product was purified via flash columnchromatography eluting with 1% methanol in dichloromethane and graduallyincreasing to 10% methanol in dichloromethane to yield a white solid(838.2 mg, 13.13%). ¹H NMR (300 MHz, MeOD) δ 1.33-1.47 (m, 246H, CH₂,CH₃), 2.03 (d, 48H, J=65.3 Hz, CH₂), 3.15 (td, 30H, J=6.3 Hz, J=12.7 Hz,CH₂), 3.29 (m, 45H, CH₂), 7.42 (m, 1H, ArH), 7.67 (d, 1H, J=8.2 Hz,ArH), 7.79 (d, 1H, J=8.1 Hz, ArH), 8.02 (s, 1H, ArH).

71. Cleavage of Disulfide Bridge on Molecular Transporter.

The disulfide linker hexyl molecular transporter (257.8 mg, 61.41 μmol)in DriSolv DMF (5 mL) was stirred under argon and a solution ofDL-dithiothreitol (740.0 mg, 4.80 mmol) in DMF (5 mL) was added dropwiseand the reaction proceeded for 2 h at room temperature. After removal ofDMF in vacuo, the reaction was purified using a Sephadex LH-20 column,eluting with DMF and concentrating the fractions in vacuo again yieldingthe product (251.0 mg, 100%).

72. Attachment of Molecular Transporter to Nanoparticles.

The nanoparticles (147.4 mg, 3.07 μmol) in DriSolv DMF (10.0 mL) werestirred under argon and the free thiol hexyl molecular transporter(251.0 mg, 61.41 μmol) in DriSolv DMF (10.0 mL) was added dropwisefollowed by the addition of a catalytic amount of N-methylmorpholine.After removal of DMF in vacuo, the product was dissolved in methanol anddialyzed against a 1:1 methanol:water solution, eventually dialyzingagainst pure methanol with SnakeSkin® Pleated Dialysis Tubing(MWCO=10,000). ¹H NMR (400 MHz, MeOD) δ 7.55-6.21 (br m, aromatic fromcrosslinker), 3.85-3.49 (br t, maleimide linker), 3.22-3.00 (br m,N-Tfa-ethylenediamine and molecular transporter), 3.00-2.70 (br m,backbone, N-Tfa-ethylenediamine, and molecular transporter), 2.70-1.00(br m, backbone and molecular transporter).

73. Deprotection of Trifluoroacetyl Protected Amines on ModifiedParticles.

The nanoparticles (142.0 mg, 1.54 μmol) were dissolved in methanol (5.0mL) and a 10% K2CO3 solution of 5:3 methanol:water (13.0 mL) was addedto the solution and the reaction proceeded overnight at roomtemperature. The reaction was purified by dialysis with SnakeSkin®Pleated Dialysis Tubing (MWCO=10 000) against a 5:3 methanol:watersolution, eventually dialyzing against pure methanol then dialyzingagainst a 1:1 methanol THF solution, eventually dialyzing against pureTHF. 1H NMR (400 MHz, THF d8) δ 8.26-6.53 (br m, aromatic fromcrosslinker), 3.94-3.52 (br m, maleimide linker), 3.28-3.12 (br t,ethylenediamine and molecular transporter), 3.12-2.68 (br m, backbone,ethylenediamine, and molecular transporter), 2.68-1.05 (br m, backboneand molecular transporter).

74. Attachment of 3-(pyridine-2-yl disulfanyl)propanoic AcidNanoparticles.

A solution of 3-(pyridine-2-yl disulfanyl)propanoic acid (16.8 mg, 77.9μmol) in anhydrous THF (2.5 mL) was stirred under argon at 0° C. withN-methylmorpholine (7.88 mg, 77.9 μmol) followed by dropwise addition ofisobutyl chloroformate (11.7 mg, 85.7 μmol). After 1.5 h, a solution ofthe deprotected nanoparticles (111.0 mg, 1.30 μmol) in anhydrous THF(35.0 mL) was added dropwise. The reaction was allowed to warm to roomtemperature and stirred for 24 h. The reaction was diluted and purifiedby dialysis with SnakeSkin® Pleated Dialysis Tubing (MWCO=10,000)against methanol, eventually dialyzing against a 3:1 THF:MeOH solution.¹H NMR (400 MHz, THF ds) δ 7.58-6.22 (br m, aromatic from crosslinkingand disulfide linker), 3.87-3.67 (br m, maleimide linker), 3.24-3.16 (brm, disulfide linker), 3.15-3.04 (br m, diamine and moleculartransporter), 2.93-2.83 (br m, diamine and molecular transporter),2.78-2.62 (br m, disulfide linker), 2.62-1.06 (br m, backbone).

75. Attachment of Alexa Fluor® 568.

To a solution of multifunctional nanoparticles (10.0 mg, 106.0 nmol) inDriSol v DMF (3.0 mL), a solution of Alexa Fluor® 568 (3.78 mg, 4.77μmol) in anhydrous DMSO (377.7 μL) and triethylamine (50.0 μL, 358.7μmol) was added to the solution and the reaction proceeded in the darkfor 24 h at room temperature. The reaction was diluted with THF an dpurified by dialysis with SnakeSkin® Pleated Dialysis Tubing(MWCO=10,000) against 1% H₂O in THF eventually dialyzing against pureTHF.

76. Capping of the Remaining Amines.

Upon completion of the Alexa Fluor 568 addition to the nanoparticles, asolution of N-acetoxysuccinimide (47.1 mg, 299.5 μmol) in DriSolv DMF(1.0 mL) was added to the reaction solution. The reaction was allowed toproceed for 3 h at RT. After removal of DMF in vacuo, the product wasdissolved in methanol and dialyzed against methanol with SnakeSkin®Pleated Dialysis Tubing (MWCO=10,000). ¹H NMR (400 MHz, MeOD) δ1.29-2.43 (br m, backbone and molecular transporter), 2.59-2.83 (br m,disulfide linker), 2.84-2.95 (br m, disulfide linker), 2.98-3.02 (br m,disulfide linker), 3.04-3.09 (br m, disulfide linker), 3.16 (br t,diamine and molecular transporter), 3.67 (br t, maleimide linker),6.53-7.98 (br m, aromatic from crosslinking, disulfide linker, andFITC).

77. Boc Deprotection of Modified Nanoparticles.

Modified nanoparticles (30.0 mg, 434.0 nmol) were dissolved in anhydrous1,4-dioxane (10 mL) and chilled to 0° C. A solution of 4 M HCl in1,4-dioxane (10 mL) was added dropwise to the stirring nanoparticles andthe reaction was allowed to proceed overnight at room temperature Thenanoparticle solution was diluted to three times the original volumewith water and dialyzed against water with SnakeSkin® Pleated DialysisTubing (MWCO=10,000). Upon completion of dialysis, the aqueous solutionwas lyophilized to yield a yellow solid. 1H NMR (400 MHz, D2O) δ1.18-2.37 (br m, backbone and molecular transporter), 2.71-2.79 (br m,disulfide linker), 2.81-2.86 (br m, disulfide linker), 2.89-2.93 (br m,disulfide linker), 2.94-2.99 (br m, disulfide linker), 3.12 (br t,diamine and molecular transporter), 3.69 (br t, maleimide linker),6.53-8.41 (br m, aromatic from crosslinking, disulfide linker, andFITC).

78. Synthesis of Copolymer poly(vl-avl) (Ab).

A 50 mL 3-necked round bottom flask, equipped with stir bar, was sealedwith two septa and a gas inlet. The flask was evacuated and refilledwith nitrogen three times. Stock solutions of 1.7 M ethanol (EtOH) inTHF and 3.7×10⁻² M tin(II) 2-ethylhexanoate (Sn(Oct)₂) in THF were madein sealed N₂ purged flasks. Solutions of EtOH (0.32 mL, 5.41×10⁻¹ mmol)and Sn(Oct)₂ (0.30 mL, 1.12×10⁻² mmol) were combined in the nitrogenpurged 50 mL flask. After stirring the mixture for 30 min,α-allyl-δ-valerolactone (1.16 g, 8.32 mmol) and δ-valerolactone (vl,2.50 g, 24.97 mmol) were added. The reaction vessel stirred at 105° C.for 48 h. Residual monomer and catalyst were removed by dialyzing withSpectra/Por® dialysis membrane (MWCO=1000) against CH₂Cl₂ to give agolden brown polymer. Yield: 3.24 g (88%). M_(w)=3042 Da, PDI=1.18; ¹HNMR (300 MHz, CDCl₃/TMS, ppm) δ: 5.7 (m, H₂C═CH—), 5.09 (m, H₂C═CH—),4.09 (m, —CH₂—O—), 3.65 (m, CH₃CH₂O—), 2.35 (m, vl, —CH₂CH₂C(O)O—, avl,H₂C═CHCH₂CH—, H₂C═CHCH₂CH—), 1.68 (m, avl & vl, —CHCH₂CH₂—), 1.25 (t,CH₃CH₂O—); ¹³C NMR (400 MHz, CDCl₃, ppm) δ: 174.6 (avl, —C(O)—), 172.7(vl, —C(O)—), 134.6 (H₂C═CH—), 116.4 (H₂C═CH—), 63.3, 44.3, 35.9, 33.1,27.5, 25.9, 23.6, 20.9.

79. Nanoparticle Formation from Ab.

A solution of Ab (0.0804 g, M_(w)=3042 Da, PDI=1.18) dissolved in CH₂Cl₂(0.18 mL) was added to a solution of 3,6-dioxa-1,8-octanedithiol (30.0μL, 0.18 mmol) in CH₂Cl₂ (28.4 mL) at 44° C. The reaction mixture washeated for 12 h. Residual dithiol was removed by dialyzing withSnakeSkin® Pleated Dialysis Tubing (MWCO=10,000) againstdichloromethane. Yield: 0.078 g. ¹H NMR (300 MHz, CDCl₃/TMS) δ: Thesignificant change is the reduction of the allyl protons at 5.06 and5.77 ppm and the appearance of signals at 3.65 and 2.71 ppmcorresponding to the protons neighboring the thiols of the PEG linkerafter cross-linking. All other aspects of the spectrum are similar tothat of Ab. The reaction can also be conducted with photoinitiators atRT in organic solvents. The particle sizes of the resulting particlescorrespond to those produced in analogous epoxide/amine procedures.

When reaction times are increased to from about 24 h to about 48 h, theparticle sizes increase due to the total consumption of allyl moieties.Reaction at room temperature was found to be sufficient. Addition ofradical starters or other photoinitiators does not significantlyincrease the quality of the particles.

80. One Pot Synthesis of Nanoparticles from poly(vl-evl-avl-opd) (ABbD).

In a 25 mL three-necked round bottom flask equipped with stir bar,condenser and septa, 2,2′-(ethylenedioxy)diethylamine (18.3 μL,1.25×10−4 mol), 17.1 mL CH₂Cl₂ and a solution of poly(vl-evl-were added.A solution of poly(vl-evl-avl-opd), ABbD, (0.0781 g, M_(w)=3500 Da,PDI=1.29). The mixture was heated at 44° C. for 12 h. Residual diaminewas removed by dialyzing with SnakeSkin® Pleated Dialysis Tubing(MWCO=10,000) against dichloromethane. ¹H NMR (300 MHz, CDCl₃/TMS, ppm)δ: The significant change is the disappearance of the epoxide protons at2.94, 2.75 and 2.47 ppm and the appearance of signals at 3.64 and 2.97ppm corresponding to the protons neighboring the secondary amine of thePEG linker after cross-linking. All other aspects of the spectrum aresimilar. TEM analysis of the resulting nanoparticles is shown in FIG.41. The particle size distribution, with unusually narrowpolydispersity, of the resulting nanoparticles is shown in FIG. 43. Toincrease particle sizes, reaction times can be increased to from about24 h to about 48 h.

81. Uptake Experiment Protocol.

Fluorescent multifunctional nanoparticle, negative control particle,FD-1, and FD-2 uptake by mammalian cells was assessed using HeLa cells,cancer cells, grown in uncoated, 14 mm diameter Microwell, No. 1.5MatTek Dishes and a Zeiss LSM 510 META confocal microscope. HeLa cellswere grown in Dulbecco's Modified Eagle's Medium-Low Glucose (DMEM)(Sigma Aldrich) supplemented with 10% (v/v) fetal bovine serum (Gibco)and 1% (v/v) antibiotic-antimycotic (Gibco). The cells were treated withthe multifunctional nanoparticles, negative control particles, FD-1, orFD-2 for one hour, washed three times with Ca²⁺/Mg²⁺ free PhosphateBuffered Saline with EDTA (PBS), fixed with 3.3% paraformaldehyde atroom temperature for 10 minutes, and analyzed using confocal microscopy.

82. Bioconjugate Molecular Transporter.

To prepare an exemplary antibody conjugated molecular transporter, aG1-Newkome dendrimer that contains nine t-butyl end functionalities anda primary amine group at the focal point was prepared using disclosedmethods (FIG. 44). The amine functionality was reacted with3-(2-pyridinyldithio)propanoic acid via amide coupling reactions withDCC/HOBt to form a protected dendimer with a reactive core. The t-butylester groups on the periphery of the dendritic scaffold were deprotectedwith formic acid to give free carboxylic acid groups that were coupledwith N-Boc-1,6-diaminohexane. After deprotection of the Boc protectinggroups with 2M HCl in dioxane the free amines were transformed intoguanidine groups with N,N-diBoc-N-triflylguanidine and the subsequentdeprotection of the Boc groups using 2 M HCl in dioxane gave the desiredcompound (Scheme 21).

The final compound is designed to localize in the cytoplasm of thecells, as an integrated a hexyl alkyl spacer is present at the peripheryof the dendrimer scaffold that was previously found to be a feature forthe specificity of its subcellular location. Furthermore, thepyidinydirectthio linker at the focal point allows for the exchange withsulfhydryl groups to form bioconjugates that are connected over adisulfide bond to afford a reductive cleavable linker that can maintainactivity of the biomolecule in cells. The IgG molecular transporterconjugate (IgGMT) was formed by the mild reaction of Alexa Fluor® 568labeled IgG antibody in PBS buffer at RT with the dendrimer (FIG. 44).Five transporter dendrimers were attached to the IgG structure which hasa molecular weight of 148 kDa. The conjugate was dialyzed against PBSbuffer to remove any unreacted dendrons and the concentration in thedialysis tubing was choosen to be 1 mg ml⁻¹ IgGMT, that allowed for theuse of the solution directly for the uptake and neutralizationexperiments.

First, the uptake efficiency of the IgGMT conjugate into humanepithelial cells (HEp-2) was tested. The 60% confluently grown cellswere incubated with IgGMT initially for 10 min then for 30 min, 1, 2 and6 h. The uptake efficiency was studied with confocal microscopy and theuptake of the bioconjugate could be observed as early as 10 min. Latertime points showed an increase of red fluorescence of the IgGMTconjugate, progressing from the cell surface membrane to localizeintracellularly in the perinuclear area at time points of 2 and 6 h.Contrary to the affinity and uptake of the IgGMT bioconjugate, the AlexaFluor® 568 labeled, unmodified IgG did not enter the cell at all timespoints investigated (FIG. 45). After the uptake into HEp-2 cells wasconfirmed with no evidence of cellular damage, the activity of theconjugate in RSV infected cells that expressed green fluorescent proteinGFP as a result of RSV infection was examined. First, HEp-2 cells wereinfected for 24 h with recombinant RSV-GFP, washed and allowed toincubate for an additional 48 h. The cells were then imaged withconfocal microscopy at a total of 72 h after initial infection (FIG.46). The typical syncytia formation was observed, a combination andfusion of the infected cells, together with the expression of the greenfluorescent protein (GFP). To study the neutralization effect of theIgGMT, HEp-2 cells infected for 24 h with RSV-GFP were incubated for 30min with a solution of IgGMT in PBS buffer, washed and imaged 48 h later(FIGS. 46a and b ). By confocal microscopy it was observed that asignificant reduction of the green fluorescence of GFP (a) and a strongred fluorescence of the Alexa Fluor® 568 labeled IgGMT conjugate (b).The merged images of (a) and (b) combined with differential interferencecontrast (DIC) also gave evidence of healthier cells with significantlyless syncytia formation than the untreated infected cells at the sametime period (FIG. 46c ). This result illustrated the significantreduction of GFP in treated cells in contrast to the untreated cellsobserved at a total incubation time of 72 h after infection with RSV for24 h. Besides the presence of neutralized cells that showed only the redfluorescence of the conjugate, cells that showed the coexsistance ofRSV-GFP and the red fluorescence of the IgGMT, appearing in the centerof the cells (FIG. 46 and FIG. 47, a+b merged) were also observed.Imaging directly after the 30 min incubation of the RSV infected cellswith the IgGMT, showed the green fluorescence localized intracellularly,whereas the red fluorescence was observed on the cell-surface membranes(FIG. 47, a+b merged). These images documented the high affinity of theconjugate with the cells surface directly after the incubation periodfollowed by the uptake into the Hep-2 cells after an additionalincubation time of 48 h. Parallel investigations of the RSV titres ofthe supernatant showed a significant reduction by 80-90% in viralreplication when compared to cells not exposed to the IgGMT conjugate.Without wishing to be bound by theory, the intracellular delivery of IgGantibody directed to the surface protein inhibits the syncytialformation mediated by the F protein and has an effect on total virusproduction when added 24 h after the initiation of RSV infection.

83. Tailored Polyester Nanoparticles.

In this example, polyester nanoparticles in controlled nanoscopicdimensions have been prepared through a one-pot procedure that containsamine, keto, and allyl groups and is tailored towards the conjugation ofbioactive building blocks, such as a dendritic molecular transporter tofacilitate cellular uptake, or peptides and dyes to accomplish targetingand imaging. In several examples of bioconjugate synthesis, demonstratedis the versatility and the orthogonal attachment strategies involvinghigh yielding thiol-ene reactions under mild conditions and reductiveamination reactions, circumventing the integration of linker andmulti-step post-modification pathways. Several linear nanoparticleprecursors were prepared according to Scheme 22.

After the collapse of the above linear precursors using disclosedmethods, the nanoparticle can be functionalized with a desired moiety.In contrast to reported strategies that form amide bonds with polyesterscaffolds using EDC activation that are typically not very high yieldingand require a high excess of expensive peptides, in this example theN-terminus of the targeting unit (e.g., a peptide) was reacted with theketo group, integrated in the polymer backbone of the developedpolyester particle. In a model reaction, the successful reductiveamination of N-Boc-ethylenediamine with keto groups of the particle hasbeen shown and applied these reaction conditions to test the addition ofpeptidic units. Here, the targeting peptide sequence GCGGGNHVGGS SV wastested and chosen for the reaction with the ABD nanoparticle, with thenanoscopic dimension of 118 nm prepared from the ABD linear precursorpolymer with 1.5 equivalents of 2,2′-(ethylenedioxy)bis(ethylamine)cross-linking units with the conditions as described above (Scheme 22).The amine groups of the nanoparticle were first capped withN-acetoxysuccinimide and the modified nanoparticle and the peptide weresolubilized in tetrahydrofuran with NaCNBH3 as the reducing reagent.

After purification through dialysis the modified particles, 3, werecharacterized with ¹H NMR and DLS. The increase in hydrodynamic diameterfrom 118±10 nm to 120±10 nm indicated the addition of peptides to thepolyester backbone and further investigations with 1H NMR showed theconjugation of peptides with the characteristic resonance peaks at 4.39and 7.42 ppm. With additional analysis through static light scattering(SLS) we could determine the amount of peptide attached to thenanoparticle that was estimated to be between 36 of the intendedattachment of 40 peptides per particle.

This result confirmed the efficiency of the reductive aminationreactions with the N-terminus of the selected peptidic units. Targetingunits, however, that contain more than one amine group give mixedconjugation products and an alternative strategy has to be developed.For this reason, we wanted to pursue thiol-ene type reactions that willbe performed between cysteine units, integrated into the sequence closeto the N-terminus, and double bonds that we find in maleiimides,vinylsulfones or allyl groups. To integrate the reaction partner for thethiol/cysteine containing units, such as peptides, or oligonucleotides,into the nanoparticles, either a suitable linker that would be attachedto the prepared nanoparticle was synthesized or a method that wouldcircumvent the conjugation of a linker molecule to facilitate theattachment of thiol containing entities was found. Therefore, theintegration of allyl groups in the polyester backbone as pendantfunctional units that would be already present in the linear polyesterprecursor before nanoparticle formation was studied. The available allylgroups that stem from the α-allyl-δ-valerolactone of the linearpolyester precursors were oxidized and converted entirely into epoxidegroups to provide units that would cross-link with the diamine. However,with partial oxidation of the allyl group, linear polyester precursorscontaining epoxide units and remaining allyl groups, could beaccomplished. In the next step, a linear polyester AbD that waspartially oxidized to comprise 16% of allyl units and 11% of epoxideunits was cross-linked with 1.5 equivalents of diamine, using the novelone-pot reaction procedure to examine the compatibility of the allylgroups to the conditions of nanoparticle formation. The investigation ofthe resulting particles with DLS showed that hydrodynamic diameterscorresponded to the size and solubility of the particles that did notcontain any allyl groups. The allyl resonance peaks were still presentin the ¹H NMR spectra of the particles and were found to be analogous tothe resonances of the allyl functionalities in the linear precursor.

After attaching a fluorescent probe, a disclosed cyclic peptide wasattached, as shown in Scheme 23 in FIG. 77.

In the next step, a combined dendritic, peptidic, nanoparticle scaffoldwas synthesized according to Scheme 24 in FIG. 78.

For the first approach, linear peptides GCGGGNHVGGSSV with therecognition unit HVGGSSV with protected amines after capping withN-acetoxysuccinimide, were conjugated to the allyl functionality of aABbD nanoparticle of 126.6 nm through the thiol of the cysteine unit asdiscussed above. In a following reaction, the imaging reagent AlexaFluor®594 was introduced to label around 20 of the incorporated amineunits of the nanoparticle. In a sequential thiolene reaction, theconjugation of 30 dendritic transporter molecules was achieved (Scheme7), as was confirmed via ¹H NMR spectroscopy. The sequential conjugationof the bioactive compounds can be followed with an overlay of the ¹H NMRspectra that show the addition of first the peptide and the remainingallyl groups of the nanoparticle and the characteristic peaks of themolecular transporter molecule at 2.0 and 3.2 ppm.

The reaction sequence was changed to obtain a similar bioconjugateproduct that was only differentiated by the peptidic targeting unit. Theamine groups of the c-RGD unit were not capped to avoid inactivation ofthe Arginine® recognition unit. Therefore the conjugation strategyincluded that the amine groups of the nanoparticle were first labeledwith the NHS Alex Fluor dye followed by the thiol-ene reaction with thetargeting unit as shown in Scheme 6. In the last step, same as in theprevious reaction, the dendritic transporter unit was added in asequential thiol-ene reaction (Scheme 25 in FIG. 79).

In a third and last reaction sequence, we could demonstrate theversatility of the provided functional units of the nanoparticle andproceeded with an orthogonal conjugation approach. The free amine groupsof the nanoparticle are capped with N-acetoxysuccinimide to notinterfere with the following reductive amination reaction between theketo group of the polyester backbone and the N-terminus of theunmodified targeting peptide HVGGSSV. After the reductive aminationreaction was completed in the same fashion as described for compound 3,a thiolene reaction between the allyl groups of the nanoparticle and thethiol group of the molecular transporter could achieve the attachment of30 units according to 1H NMR spectroscopy analysis. The additional finalcharacterization of the modified particles with static light scattering(SLS) the number of conjugated peptides peptides could determine theaddition of 36 peptides to the particle. In a last step, the NHS esterAlexa Fluor dye was modified with thiolethylamine (Scheme 26 in FIG. 80)to label exclusively the particle through a thiol-ene reaction to imagethe system in vitro. The Alexa Fluor 594 dye proved to be stable underthe conditions and another example of the chemical versatility of thesystem was given.

TABLE 6 Summary of nanoparticle conjugates with definition of particletype depending on linear polymer precursora and connected targetingpeptideb: ‘c’ for capped N-terminus of peptide with HVGGSSV recognitionunit via N-acetoxysuccinimide and ‘c’ for cyclic RGD. Alexa DendriticParticle Targeting Fluor ® Molecular Compound Type^(a) Peptides^(b) DyeTransporter^(c) Compound Name^(d) Class ABD HVGGSSV — — ABD-NP-HVGGSSV(3) NP-P ABbD HVGGSSV — — ABbD-NP-HVGGSSV (14) NP-P ABbD cHVGGSSV 594 —ABbD-NP-cHVGGSSV-594 (8) NP-P-dye ABbD cRGD 594 — ABbD-NP-594-cRGD (10)NP-P-dye ABbD — 594 MT ABbD-NP-594-MT (6) NP-MT-dye ABbD cHVGGSSV 594 MTABbD-NP-cHVGGSSV-594-MT(11) NP-P-MT-dye ABbD cRGD 594 MTABbD-NP-594-cRGD-MT (12) NP-P-MT-dye ABbD HVGGSSV 594 MT ABbD-NP-594-MT(16) NP-P-MT-dye ^(c)Dendritic molecular transporter is abbreviated asMT, and the compound name is given in the order of the attachment d.

Below are the experimental procedures relevant to Example 123.

Synthesis of Copolymer poly(vl-avl-opd) (AbD).

To a 25 mL 3-necked round bottom flask, equipped with stir bar, gasinlet and 2 rubber septa, 2-oxepane-1,5-dione (0.70 g, 5.46 mmol) wasadded. The round bottom flask was purged with argon. After purging for30 min, dry toluene (4 mL) was added. The mixture stirred in an oil bathat 80° C. to dissolve the monomer. Upon dissolving, Sn(Oct)₂ (11.1 mg,27.3 μmol) in 0.5 mL dry toluene, absolute ethanol (20.5 mg, 440 μmol),α-allyl-δ-valerolactone (1.15 g, 8.19 mmol) and δ-valerolactone (1.37 g,13.7 mmol) were then added to the reactor and the mixture was heated for48 h at 105° C. Residual monomer and catalyst were removed by dialyzingwith Spectra/Por® dialysis membrane (MWCO=1000) against CH2Cl2 to give agolden brown polymer. Yield: 2.70 g (85%). Mw=3287 Da, PDI=1.17; ¹H NMR(300 MHz, CDCl3/TMS, ppm) δ: 5.72 (m, H2C═CH—), 5.06 (m, H2C═CH—), 4.34(m, —CH2CH2C(O)CH2CH2O—), 4.08 (m, —CH2O—), 3.67 (m, —OCH2CH3), 2.78 (m,opd, —OC(O)CH2CH2C(O)CH2-), 2.58 (m, opd, —OC(O)CH2CH2C(O)CH2-), 2.34(m, vl, —CH2CH2C(O)O—, avl, H2C═CHCH2CH—, H2C═CHCH2CH—), 1.66 (m, avl &vl, —CHCH2CH2-), 1.25 (t, —CH2CH3); 13C NMR (400 MHz, CDCl3, ppm) δ:204.9, 175.2, 173.7, 173.2, 135.0, 117.0, 63.9, 44.8, 36.4, 33.6, 28.0,26.3, 21.3.

Synthesis of poly(vl-evl-opd) (ABD).

To a solution of AbD (2.70 g, 4.67 mmol) in CH2Cl2 (37 mL),3-chloroperoxybenzoic acid (1.46 g, 8.48 mmol) was added. The mixturestirred for 72 h at room temperature and then concentrated via rotaryevaporator. The crude product was dissolved in a minimal amount oftetrahydrofuran (THF) (5 mL) and dropped into a round bottom flaskcontaining 1 L diethyl ether. The solution was kept overnight at 0° C.and a white solid was obtained. The solution was decanted off and thesolid was dried in vacuo to obtain ABD. Yield: 1.95 g (72%). Mw=3392 Da,PDI=1.19. 1H NMR (300 MHz, CDCl3/TMS) δ: The significant change is thedisappearance of the allylic protons at 5.74 and 5.09 ppm and theappearance of small broad resonance peaks at 2.94, 2.75 and 2.47 ppm dueto the formation of the epoxide ring. All other aspects of the spectrumare similar.

Nanoparticle Formation from poly(vl-evl-opd) (ABD).

A solution of ABD (0.11 g, Mw=3392 Da, PDI=1.19) dissolved in CH2Cl2(0.26 mL) was added dropwise via a peristaltic pump at 13 mL/min withvigorous stirring to a solution of 2,2′-(ethylenedioxy)diethylamine(76.4 μL, 0.52 μmol) in CH2Cl2 (40.3 mL) at 44° C. The mixture washeated for 12 h. Residual diamine was removed by dialyzing withSnakeSkin® Pleated Dialysis Tubing (MWCO=10,000) againstdichloromethane. Yield: 0.17 g (91%). DLS:DH=118.3±9.6 nm. SLS:Mw=323,000. 1H NMR (300 MHz, CDCl3/TMS) δ: The significant change is thedisappearance of the epoxide protons at 2.94, 2.75 and 2.47 ppm and theappearance of signals at 3.54 and 2.97 ppm corresponding to the protonsneighboring the secondary amine of the PEG linker after cross-linking.All other aspects of the spectrum are similar.

N-Boc-Ethylenediamine (NBED) Conjugated ABD Nanoparticles.

To a solution of ABD nanoparticles (20 mg, 0.06 μmol) in THF (2 mL),N-acetoxysuccinimide (0.02 g, 0.13 mmol) was added. The reaction mixturestirred for 3 h. Residual N-acetoxysuccinimide was removed by dialyzingwith SnakeSkin® Pleated Dialysis Tubing (MWCO=10,000) against THF. Oncethe product was concentrated and dried, the nanoparticles (18 mg, 0.05μmol) were dissolved in a mixture of CH2Cl2 and CH3OH (1:1, v/v, 2 mL).To this solution, N-Boc-ethylenediamine (4.6 μL of 1.59 M NBED in CH3OH)and NaCNBH3 (21.8 μL of 1.0 M NaCNBH3 in THF) were added. The reactionmixture stirred for 12 h at room temperature and then was purified bydialyzing with SnakeSkin® Pleated Dialysis Tubing (MWCO=10,000) against1:1 CH2Cl2/CH3OH. Yield: 18 mg (88%). DLS:DH=119.5±10.3 nm; originalparticle DH=118.3±9.6 nm. 1H NMR (300 MHz, CDCl3/TMS) δ: The significantchange is the appearance of the peak at 1.43 ppm due to the Bocprotecting group. All other aspects of the spectrum are similar to thatof the ABD nanoparticles.

General Procedures for the Synthesis of HVGGSSV Peptide (1).

The HVGGSSV peptide was synthesized by solid-phase peptide synthesisusing standard Fmoc chemistry on a Model 90 Peptide Synthesizer(Advanced ChemTech). General procedure: Attachment of N-Fmoc amino acidsto resin. After swelling with dichloromethane (20 mL) for 20 min,H-val-2-Cl-Trt resin (0.20 g, 1.03 mmol/g, 0.21 mmol surface aminoacids) was treated with a solution of Fmoc-protected amino acids (4.4equiv, 0.9 mmol) in dimethylformamide (DMF) (9 mL). The amino acids wereattached to the resin using double coupling with a solution (9 mL)consisting of N-hydroxybenzotriazole monohydrate (HOBt) (0.9 mmol, 0.14g) o-(benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate(0.9 mmol, 0.34 g), N,N′-diisopropylethylamine (DIPEA) (1.8 mmol, 0.31mL) in 9 mL DMF. The reaction mixture was shaken for 60 min and washedwith DMF (4×10 mL), methanol (4×10 mL) and DMF (4×10 mL). The end of thecoupling was controlled by the Ninhydrin test. A 20% (v/v) piperidine inDMF solution was used to deprotect the Fmoc groups. The amino acids wereattached to the resin in the following sequence: Ser, Ser, Gly, Gly,Val, His, Asn, Gly, Gly, Gly, Cys, and Gly.

General Procedure: Cleavage from Resin.

The resin was treated with Reagent R, a solution of TFA, thioanisole,anisole, and ethanedithiol (90:5:3:2, 6 mL), for 4 h. After removal ofthe resin by filtration, the filtrate was concentrated to precipitatethe peptide with cold diethyl ether. Crude peptides were purified byRP-HPLC and lyophilized. Peptide identity was confirmed by MALDI-MS(m/z: 1087.1).

HVGGSSV Conjugated ABD Nanoparticles (3).

To a solution of ABD nanoparticles (20.0 mg, 0.06 μmol) in THF (2 mL),N-acetoxysuccinimide (3 mg, 18.1 μmol) was added. The reaction mixturestirred for 3 h. Residual N-acetoxysuccinimide was removed by dialyzingwith SnakeSkin® Pleated Dialysis Tubing (MWCO=10,000) against 1:1THF/CH3OH to give amine capped ABD nanoparticles, 2. To a solution of 2(0.0174 g, 0.05 μmol, in 3 mL THF), 1 (3.5 mg, 3.18 μmol) dissolved inDMSO (2 mL) and NaCNBH3 (6.36 μL 1.0 M NaCNBH3 in THF) were added. Thereaction mixture stirred for 12 h at room temperature. The reactionmixture was purified by dialyzing with SnakeSkin® Pleated DialysisTubing (MWCO=10,000) against 1:1 THF/CH3CN. Yield: 19 mg (88%)DLS:DH=120.5±10.2 nm; original particle DH=118.3±9.6 nm. SLS:Mw=362,000; original particle Mw=323,000. 1H NMR (600 MHz, (CD3)2SO) δ:The significant change is the appearance of the following peaks:8.26-7.87, 7.42, 6.90, 4.39, and 4.25 ppm due to the attachment of thepeptide. All other aspects of the spectrum are similar to that of theABD nanoparticles.

Synthesis of poly(vl-evl-avl-opd) (ABbD).

To a solution of AbD (1.70 g, 1.56 mmol) in CH2Cl2 (30 mL),3-chloroperoxybenzoic acid (0.22 g, 1.28 mmol) was added. The mixturestirred for 72 h at room temperature and then was concentrated viarotary evaporator. The crude product was dissolved in a minimal amountof THF (5 mL) and poured into a round bottom flask containing 1 Ldiethyl ether. The solution was kept overnight at 0° □C. and a whitesolid was obtained. The solution was decanted off and the solid wasdried in vacuo to obtain ABbD. Yield: 1.2 g (71%). Mw=3356 Da, PDI=1.18.1H NMR (300 MHz, CDCl3/TMS, ppm) δ: 5.72 (m, H2C═CH—), 5.06 (m,H2C═CH—), 4.34 (m, —CH2CH2C(O)CH2CH2O—), 4.08 (m, —CH2O—), 3.67 (m,—OCH2CH3), 2.96 (m, epoxide proton), 2.78 (m, evl epoxide proton, opd,—OC(O)CH2CH2C(O)CH2-), 2.58 (m, opd, —OC(O)CH2CH2C(O)CH2-), 2.47(epoxide proton), 2.34 (m, vl, —CH2CH2C(O)O—, avl, H2C═CHCH2CH—,H2C═CHCH2CH—), 1.66 (m, avl & vl, —CHCH2CH2-), 1.25 (t, —CH2CH3).

Nanoparticle formation from ABbD.

A solution of ABbD (0.21 g, Mw=3356 Da, PDI=1.18) dissolved in CH2Cl2(0.39 mL) was added dropwise via a peristaltic pump at 13 mL/min withvigorous stirring to a solution of 2,2′-(ethylenedioxy)diethylamine(42.6 μL, 0.29 mmol) in CH2Cl2 (60 mL) at 44° C. The reaction mixturewas heated for 12 h. Residual diamine was removed by dialyzing withSnakeSkin® Pleated Dialysis Tubing (MWCO=10,000) againstdichloromethane. Yield: 0.24 g (96%). DLS:DH=123.4±9.22 nm. SLS:Mw=345,000. 1H NMR (300 MHz, CDCl3/TMS) δ: The significant change is thedisappearance of the epoxide protons at 2.96, 2.75 and 2.47 ppm and theappearance of signals at 3.56 and 2.98 ppm corresponding to the protonsneighboring the secondary amine of the PEG linker after crosslinking.All other aspects of the spectrum are similar to that of ABbD.

One Pot Synthesis of Nanoparticles from ABbD.

To a solution of 2,2′ (ethylenedioxy)diethylamine (26.2 μL, 0.18 mmol)in CH2Cl2 (34.6 mL), a solution of ABbD (0.13 g, Mw=3356 Da, PDI=1.18)in CH2Cl2 (0.24 mL) was added. The mixture was heated at 44° C. for 12h. Residual diamine was removed by dialyzing with SnakeSkin® PleatedDialysis Tubing (MWCO=10,000) against CH2Cl2. Yield: 0.15 g (94%).DLS:DH=126.6±9.3 nm. SLS: Mw=350,000. 1H NMR (300 MHz, CDCl3/TMS) δ: Thesignificant change is the disappearance of the epoxide protons at 2.94,2.75 and 2.47 ppm and the appearance of signals at 3.54 and 2.97 ppmcorresponding to the protons neighboring the secondary amine of the PEGlinker after cross-linking. All other aspects of the spectrum aresimilar to that of ABbD.

General Procedure for the Attachment of Benzyl Mercaptan to ABbDNanoparticles.

To a solution of ABbD nanoparticles (15 mg, 0.04 μmol) in toluene (0.5mL), benzyl mercaptan (3.5 μL, 29 μmol) was added. The reaction mixturewas heated for 72 h at 35° C. The remaining toluene was removed in vacuoand residual benzyl mercaptan was removed by dialyzing with SnakeSkin®Pleated Dialysis Tubing (MWCO=10,000) against CH2Cl2. 1H NMR (300 MHz,CDCl3/TMS) δ: The significant change is the reduction of the allylprotons at 5.72 and 5.06 ppm and the appearance of signals at 3.73 and7.30 ppm corresponding to the methylene and benzene protons respectivelyof the attached benzyl mercaptan. All other aspects of the spectrum aresimilar to that of ABbD nanoparticles.

Deprotection of molecular transporter (MT) (5) (contribution of SharonHamilton). To a solution of LL-MT (15 mg, 4.56 μmol) in CH3OH (0.4 mL),a solution of D,L-dithiothreitol in CH3OH (0.2 mL) was added. Thereaction mixture stirred for 3 h at room temperature. Residualdithiothreitol was removed by purification with Sephadex LH-20. Theproduct was immediately attached to ABbD nanoparticles.

Model reaction of attachment of MT to ABbD nanoparticles. To a solutionof ABbD nanoparticles (15 mg, 0.04 μmol) in CH3OH (0.2 mL), 5 (11 mg,3.35 μmol) in CH3OH (0.4 mL) was added. The reaction mixture was heatedfor 72 h at 37° C. Residual 5 was removed by dialyzing with SnakeSkin®Pleated Dialysis Tubing (MWCO=10,000) against methanol. Yield: 31.3 mg(89%). DLS:DH=128.9±10.2 nm; original particle DH=126.6±9.3 nm. ¹H NMR(300 MHz, CD3OD) δ: The significant change is the reduction of the allylprotons at 5.72 and 5.06 ppm and the appearance of signals at 2.20-1.98(CH2), 1.57 (CH2) and 1.39 (CH2) ppm due to the dendritic backbone ofthe MT. All other aspects of the spectrum are similar to that of ABbDnanoparticles.

Alexa Fluor® 594 conjugated ABbD nanoparticles (4). To a solution ofABbD nanoparticles (0.021 g, 0.06 μmol) in dry THF (1.5 mL), AlexaFluor® 594 (0.14 mL of 10 mg/mL Alexa Fluor® 594 in DMF, 1.7 μmol) wasadded. The reaction mixture stirred for 24 h at room temperature.Residual Alexa Fluor® 594 was removed by dialyzing with SnakeSkin®Pleated Dialysis Tubing (MWCO=10,000) against CH3OH. Yield: 15.2 mg(88%). 1H NMR (300 MHz, CD3OD) δ: The significant change is theappearance of the following peaks due to Alexa Fluor® 594: 7.14-7.20,6.78, 5.48, 4.48, 3.62, 3.43, and 1.24 ppm. 1H NMR (600 MHz, (CD3)2SO)δ: The significant change is the appearance of the following peaks dueto Alexa Fluor® 594: 7.52, 7.47, 7.08, 5.32, 4.44, 4.35, 3.58, 3.16,2.03, and 1.25 ppm. All other aspects of the spectrum are similar tothat of ABbD nanoparticles.

Attachment of MT to Alexa Fluor® 594 Conjugated ABbD Nanoparticles,NP-594-MT (6).

To a solution of 4 (8 mg, 0.89 μmol) in CH3OH (0.2 mL), 5 (7.5 mg, 2.27mol) in CH3OH (0.4 mL) was added. The reaction mixture was heated for 72h at 37° C. Residual 5 was removed by dialyzing with SnakeSkin® PleatedDialysis Tubing (MWCO=10,000) against CH3OH. Yield: 10.0 mg (91%).DLS:DH=129.4±9.8 nm; original particle DH=126.6±9.3 nm. 1H NMR (300 MHz,CD3OD) δ: The significant change is the reduction of the allyl protonsat 5.72 and 5.06 ppm and the appearance of signals at 2.20-1.98 (CH2),1.57 (CH2) and 1.39 (CH2) ppm due to the dendritic backbone of the MT.All other aspects of the spectrum are similar to that of 4.

N-acetoxysuccinimide Conjugated HVGGSSV Peptide, cHVGGSSV (7).

To a solution of 1 (29.4 mg, 2.7×10−5 mol) dissolved in CH3CN (3 mL),N-acetoxysuccinimide (0.42 g, 2.7×10−3 mol) was added. The reactionmixture stirred for 3 h at room temperature. After removal of thesolvent under reduced pressure, the crude product was purified byRP-HPLC. MALDI-MS: m/z=(M±H±) 1174.2.

Capped HVGGSSV Conjugated Alexa Fluor® 594-ABbD Nanoparticles,NP-cHVGGSSV-594.

To a solution of ABbD nanoparticles (0.021 g, 0.06 μmol) indimethylsulfoxide (0.7 mL), 7 (6.4 mg, 5.46 μmol) was added. Thereaction mixture was heated for 72 h at 33° C. To this solution, AlexaFluor® 594 (0.14 mL of 10 mg/mL Alexa Fluor® 594 in DMF, 1.7 μmol) wasadded. Residual Alexa Fluor® 594 and peptide were removed by dialyzingwith SnakeSkin® Pleated Dialysis Tubing (MWCO=10,000) against 1:1CH3OH/CH3CN. Yield: 20.1 mg (80%). DLS:DH=128.9±10.9 nm; originalparticle DH=126.6±9.3 nm. 1H NMR (600 MHz, (CD3)2SO) δ: The significantchange is the reduction of the allyl protons at 5.72 and 4.97 ppm andthe appearance of the following sets of significant signals: 8.21, 7.83,4.55, 3.73 and 0.80 ppm due to the peptide, and 7.25, 7.16, 6.53, 5.32,4.44, 4.37, and 1.25 ppm due to the Alexa Fluor® 594. All other aspectsof the spectrum are similar to that of ABbD nanoparticles.

Attachment of MT to cHVGGSSV Conjugated Alexa Fluor® 594-ABbDNanoparticles, NP-cHVGGSSV-594-MT.

To a solution of 8 (6 mg, 0.02 μmol) in DMSO (0.1 mL), 5 (2 mg, 0.88μmol) in CH3OH (0.3 mL) was added. The reaction mixture was heated for48 h at 33° C. Residual 5 was removed by dialyzing with SnakeSkin®Pleated Dialysis Tubing (MWCO=10,000) against 1:1 CH3OH/CH3CN. Yield:7.4 mg (93%). DLS: DH=130.7±9.4 nm; original particle DH=126.6±9.3 nm.1H NMR (600 MHz, (CD3)2SO) δ: The significant change is the reduction ofthe allyl protons at 5.72 and 4.97 ppm and the appearance of signals at3.06 (CH2), 2.96 (CH2), 1.97 (CH2), 1.77 (CH2), 1.41 (CH2) and 1.35(CH2) ppm due to the dendritic backbone of the MT. All other aspects ofthe spectrum are similar to that of 8.

Synthesis of Cyclic RGD, cRGD (9)

The RGD peptide was synthesized by solid-phase peptide synthesis usingstandard Fmoc chemistry on a Model 90 Peptide Synthesizer (AdvancedChemTech).

Synthesis of Linear RGD.

After swelling with dichloromethane (20 mL), Fmoc-Cys-2-Cl-Trt resin(0.20 g, 0.9 mmol/g, 0.18 mmol surface amino acids) was deprotected witha 20% (v/v) piperidine in DMF solution and treated with a solution ofFmoc-protected amino acid (4.4 equiv, 0.9 mmol) in dimethylformamide(DMF) (9 mL). The amino acids were attached to the resin using doublecoupling with a solution (9 mL) consisting of N-hydroxybenzotriazolemonohydrate (0.9 mmol, 0.14 g)o-(benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (0.9mmol, 0.34 g), N,N′-diisopropylethylamine (1.8 mmol, 0.31 mL) in 9 mLDMF. The reaction mixture was shaken for 60 min and washed with DMF(4×10 mL), methanol (4×10 mL) and DMF (4×10 mL). A 20% (v/v) piperidinein DMF solution was used to deprotect the Fmoc groups. An amino-hexylspacer was coupled to the cystine on the resin, followed by glutamicacid, aspartic acid, glycine, arginine, phenylalanine, and finallylysine.

Cyclization of RGD.

The peptide was cyclized by utilizing an ODmab group, which allows forthe selective deprotection carboxylic acid side chain of the glutamicacid, which can then be coupled to the N-terminus. The ODmab wasdeprotected using 2% v/v hydrazine monohydrate/DMF added to the resinand shaken for 7 min. Next it was washed with 20 mL of DMF followed by10 mL of a 5% v/v DIPEA/DMF solution which was allowed to shake for 10min. Carboxy activation was achieved through the use ofN,N′-dicyclohexylcarboimide (DCC) (44.6 mg, 0.22 mmol) andhydroxybenzotriazole (HOBt) (29.2 mg, 0.22 mmol) which was added to 10mL of DMF and then added to the resin and allowed to shake for 18 h.

General Procedure: Cleavage from Resin.

The resin was treated with Reagent R, a solution of TFA, thioanisole,anisole, and ethanedithiol (90:5:3:2, 6 mL), for 3 h. After removal ofthe resin by filtration, the filtrate was concentrated to precipitatethe peptide with cold diethyl ether. The crude peptide was collected bycentrifugation, purified by RP-HPLC and lyophilized. Peptide identitywas confirmed by MALDI-MS (m/z: 945).

Attachment of cRGD to Alexa Fluor® 594 Conjugated ABbD Nanoparticles,NP-594-cRGD (10).

To a solution of ABbD nanoparticles (23.0 mg, 0.07 μmol) in THF (2.3mL), Alexa Fluor® 594 (0.15 mL of 10 mg/mL Alexa Fluor® 594 in DMF, 1.83μmol) was added. After stirring the reaction mixture for 24 h at roomtemperature, the solvent was removed via rotary evaporator. To the AlexaFluor® 594 conjugated nanoparticles, methanol (0.35 mL) and 9 (5.7 mg,6.0 μmol), dissolved in DMSO (0.35 mL), were added. The reaction mixturewas heated for 72 h at 33° C. Residual Alexa Fluor® 594 and peptide wereremoved by dialyzing with SnakeSkin® Pleated Dialysis Tubing(MWCO=10,000) against 1:1 CH3OH/CH3CN. Yield: 22.0 mg (81%).DLS:DH=129.8±9.6 nm; original particle DH=126.6±9.3 nm. 1H NMR (600 MHz,(CD3)2SO) δ: The significant change is the reduction of the allylprotons at 5.72 and 4.97 ppm and the appearance of the following sets ofsignificant signals: 7.37, 4.79, 2.23 and 1.66 ppm due to cRGD, and7.25, 6.55, 5.31, 4.44, and 1.23 ppm due to the Alexa Fluor® 594. Allother aspects of the spectrum are similar to that of ABbD nanoparticles.

Attachment of MT to cRGD Conjugated Alexa Fluor® 594-ABbD Nanoparticles,NP-594-cRGD-MT (12).

To a solution of 10 (7.8 mg, 0.02 μmol) in DMSO (0.1 mL), 5 (1.4 mg,0.67 μmol) in CH3OH (0.3 mL) was added. The reaction mixture was heatedfor 48 h at 33° C. Residual 5 was removed by dialyzing with SnakeSkin®Pleated Dialysis Tubing (MWCO=10,000) against 1:1 CH3OH/CH3CN. Yield:7.6 mg (83%). DLS: DH=131.9±10.6 nm; original particle DH=126.6±9.3 nm.1H NMR (600 MHz, (CD3)2SO) δ: The significant change is the reduction ofthe allyl protons at 5.72 and 4.97 ppm and the appearance of signals at3.04 (CH2), 2.98 (CH2), 1.98 (CH2), 1.75 (CH2), 1.41 (CH2), and 1.35(CH2) ppm due to the dendritic backbone of the MT. All other aspects ofthe spectrum are similar to that of 11.

HVGGSSV Conjugated ABbD Nanoparticles, NP-HVGGSSV (14).

To a solution of ABbD nanoparticles (50.0 mg, 0.14 μmol) in THF (2 mL),N-acetoxysuccinimide (7 mg, 44.5 μmol) was added. The reaction mixturestirred for 3 h. Residual N-acetoxysuccinimide was removed by dialyzingwith SnakeSkin® Pleated Dialysis Tubing (MWCO=10,000) against 1:1THF/CH3OH to give amine capped ABbD nanoparticles, 13. To a solution of13 (50.0 mg, 0.14 μmol, in 3 mL THF), 1 (9.3 mg, 8.57 μmol) dissolved inDMSO (2 mL) and NaCNBH3 (17.1 μL 1.0 M NaCNBH3 in THF) were added. Thereaction mixture stirred for 12 h at room temperature. The reactionmixture was purified by dialyzing with SnakeSkin® Pleated DialysisTubing (MWCO=10,000) against 1:1 THF/CH3CN. Yield: 43.2 mg (83%).DLS:DH=129.7±9.5 nm; original particle DH=126.6±9.3 nm. SLS: Mw=391,000;original particle Mw=350,000. 1H NMR (600 MHz, (CD3)2SO, ppm) δ: Thesignificant change is the appearance of the following peaks: 8.21, 7.85,4.55, 3.73 and 0.80 ppm due to the peptide. All other aspects of thespectrum are similar to that of ABbD nanoparticles.

Thiolated Alexa Fluor® 594 (15).

To a solution of Alexa Fluor® 594 (0.2 mL of 10 mg/mL Alexa Fluor® 594in DMF, 2.4 μmol), cystemaine (68.4 μL of 2.5 mg/mL cysteamine in DMSO,2.2 μmol) was added. The reaction mixture stirred for 3 h at roomtemperature. The product was immediately attached to 14.

Attachment of MT to HVGGSSV conjugated Alexa Fluor® 594-ABbDnanoparticles, NPHVGGSSV-594-MT (16). To a solution of 14 (16 mg, 0.04μmol) in DMSO (0.2 mL), 15 (2 mg, 1.95 μmol) in DMSO (0.2 mL) and 5 (2.7mg, 1.2 μmol) in CH3OH (0.4 mL) were added. The reaction mixture washeated for 48 h at 33° C. Residual 5 and 15 were removed by dialyzingwith SnakeSkin® Pleated Dialysis Tubing (MWCO=10,000) against CH3OH.Yield: 18.5 mg (86%). DLS:DH=132.1±9.3 nm; original particleDH=126.6±9.3 nm. 1H NMR (600 MHz, (CD3)2SO) δ: The significant change isthe reduction of the allyl protons at 5.72 and 4.97 ppm and theappearance of the following sets of significant signals: 3.08, 2.99,1.97, 1.79, 1.43 and 1.34 ppm due to the dendritic backbone of the MT,and 7.27, 7.07, 6.53, 5.32, 4.46, 4.37, and 1.24 ppm due to the AlexaFluor® 594. All other aspects of the spectrum are similar to that of 14.

84. Paclitaxel Encapsulation in poly(vl-evl-avl-opd) (ABbD)Nanoparticles.

To a 150 mL beaker containing D-α-tocopherol polyethylene glycol 1000succinate (0.39 g) dissolved in Lonza cell culture water (78 mL),poly(vl-evl-avl-opd), ABbD, nanoparticles (0.17 g) and paclitaxel (34.0mg) dissolved in dimethyl sulfoxide (0.75 mL) was added slowly withvigorous stirring. The solution was split into two 50 mL centrifugetubes. The paclitaxel loaded nanoparticles were purified by applying twocycles of centrifugation (8000 rpm for 1 h) and reconstitution with cellculture water. The nanoparticle suspension was then lyophilized. Theloading ratio of paclitaxel for the encapsulation was determined byNanoDrop UV/Vis and was found to be 11.34%.

85. In Vivo Administration of Nanoparticle-Bioconjugate.

Five adult Sprague-Dawley rats were sacrificed by lethal inhalation ofCO₂. At the moment of euthanasia, eight eyes of four rats were treatedwith a solution of 2×10⁻² M nanoparticle conjugate in a molar ratio of5:1 (dye:transporter) up to 15 minutes, one rat served as the notreatment control. The solution was dropped with a micropipette on tothe cornea and multiple drops were instilled in series to maintain atear meniscus over the cornea. The rats were kept in the dark in a coldroom for two hours after the treatment and underwent encleation of theglobe with optic nerve stump attached. The eye globes with attachedoptic nerves were placed in 4% paraformaldehyde until paraffinembedding. The paraffin blocks were cut into 4-μm sections and werestained with traditional DAPI dye. Slides were viewed at 40×'smagnification using a digital fluorescent microscope Olympus Provis AX70digitally interfaced with a semi-cooled CCD camera to visualize AlexaFluor 594-labeled transporter. Background autofluorescence wassubtracted and the settings were held constant for both the control andthe treatment eyes. To proof and image the intended eye region, imagesof the same location were measured under the DAPI and Alexafluorwavelength with the microscope-mounted camera (see FIG. 50, A-D).

86. Synthesis of Copolymer poly(vl-opd).

To a 25 mL 3-necked round bottom flask, equipped with stir bar,2-oxepane-1,5-dione (0.7 g, 5.46 mmol) was added and the flask wassealed with two septa and a gas inlet. The flask was evacuated andrefilled with argon three times. Dry toluene (4 mL) was added and themixture stirred in an oil bath at 70° C. to dissolve the monomer. Upondissolving, Sn(Oct)₂ (20 mg, 5.48×10⁻² mmol in 0.5 mL dry toluene),absolute ethanol (51.1 L, 8.86×10¹ mmol), and δ-valerolactone (2.87 mL,30.7 mmol) were added. The temperature of the oil bath was increased to105° C. and the mixture stirred for 48 h. The crude product wasdissolved in a minimal amount of THF (5 mL) and poured into a roundbottom flask containing 1 L diethyl ether. The solution was keptovernight at 0° C. and a white solid was obtained. The solution wasdecanted off and the solid was dried in vacuo to obtain poly(vl-opd).Yield: 2.31 g. M_(w)=3525 Da, PDI=1.27; ¹H NMR (300 MHz, CDCl₃/TMS, ppm)δ: 4.34 (m, opd, —CH₂CH₂C(O)CH₂CH₂O—), 4.08 (m, vl, —CH₂O—), 3.65 (m,—OCH₂CH—₃), 2.74 (m, opd, —OC(O)CH₂CH₂C(O)—), 2.60 (m, opd,—CH₂CH₂C(O)CH₂CH₂—), 2.34 (m, vl, —CH₂CH₂C(O)O—), 1.68 (m, vl,—CHCH₂CH₂—), 1.25 (m, —CH₂CH₃).

87. Nanoparticle Formation from poly(vl-opd) Via Reductive Amination.

In a 100 mL round bottom flask equipped with stir bar, poly(vl-opd)(0.16 g) was dissolved in CH₂Cl₂ (11.5 mL). After dissolving thepolymer, tetrahydrofuran (11.5 mL), 2,2′-(ethylenedioxy)bisethylamine(16.9 μL, 0.12 mmol), and NaBH₃CN (1.2 mL, 1.2 mmol) were added. The pHwas adjusted to 6-7 using 1M NaOH and the reaction stirred for 12 h atroom temperature. Residual polymer, diamine and NaBH₃CN were removed bydialyzing with SnakeSkin® Pleated Dialysis Tubing (MWCO=10,000) against50/50 CH₂Cl₂/CH₃OH. ¹H NMR (300 MHz, CDCl₃/TMS, ppm) δ: The significantchange is the appearance of signals at 3.66 ppm corresponding to theprotons neighboring the secondary amine of the PEG linker aftercross-linking. All other aspects of the spectrum are similar. Theparticle size of the nanoparticles formed for various stoichiometrieswas investigated by dynamic light scattering, as tablated in Table 5,below.

TABLE 5 Size Analysis from Dynamic Light Scattering Diameter (nm)Diameter (nm) Ab₁ nanoparticles Ab₂ nanoparticles Amine/1 Keto 7% keto12% keto 2 11.3 ± 1.2 18.5 ± 1.9 3 20.7 ± 1.8 26.4 ± 2.4 4 38.1 ± 4.047.1 ± 4.9 6 77.4 ± 6.7 107.6 ± 8.9 

A example preparation of degradable polyester nanoparticle fromcopolymer poly(vl-opd) is illustrated in FIG. 51. A transmissionelectron microscopy (TEM) image of particles formed is provided in FIG.52.

88. General Nanoparticle Formation Utilizing3,6-dioxa-1,8-octanedithiol.

A solution of poly(avl-vl) (0.14 g, M_(w)=3042 Da, PDI=1.18) dissolvedin CH₂Cl₂ (0.16 mL) was added to a solution of3,6-dioxa-1,8-octanedithiol (19.6 μL, 0.12 mmol) in CH₂Cl₂ (24.6 mL).The reaction mixture was heated for 12 h at 45° C. Residual dithiol wasremoved by dialyzing with SnakeSkin Pleated Dialysis Tubing(MWCO=10,000) against CH₂Cl₂. Yield: 0.13 g. DLS: D_(H)=72.6±2.8 nm. ¹HNMR (300 MHz, CDCl₃/TMS) δ: The significant change is the reduction ofthe allyl protons at 5.06 and 5.77 ppm and the appearance of signals at3.65 and 2.71 ppm corresponding to the protons neighboring the thiols ofthe PEG linker after cross-linking. All other aspects of the spectrumare similar to that of poly(avl-vl).

89. General Nanoparticle Formation Utilizing PEG Dithiol.

A solution of poly(avl-vl) (0.13 g, M_(w)=3042 Da, PDI=1.18) dissolvedin CH₂Cl₂ (0.16 mL) was added to a solution of PEG dithiol (0.13 g, 38.1μmol) in CH₂Cl2 (23.5 mL). The reaction mixture was heated for 12 h at45° C. Residual dithiol was removed by dialyzing with SnakeSkin® PleatedDialysis Tubing (MWCO=25,000) against CH₂Cl₂. Yield: 0.11 g. DLS:D_(H)=33.7±3.8 nm. ¹H NMR (300 MHz, CDCl₃/TMS) δ: The significant changeis the reduction of the allyl protons at 5.06 and 5.77 ppm and theappearance of signals at 3.65 and 2.71 ppm corresponding to the protonsneighboring the thiols of the PEG linker after cross-linking. All otheraspects of the spectrum are similar to that of poly(avl-vl).

90. Synthesis of Copolymer poly(propargylvalerolactone-valerolactone)(poly(pvl-vl)).

A 25 mL 3-necked round bottom flask, equipped with stir bar, was sealedwith two septa and a gas inlet. The flask was evacuated and refilledwith argon three times. Stock solutions of 1.7 M ethanol (EtOH) in THFand 3.7×10⁻² M tin(II) 2-ethylhexanoate (Sn(Oct)₂) in THF were made insealed Ar_((g)) purged flasks. Solutions of EtOH (0.13 mL, 0.22 mmol)and Sn(Oct)₂ (0.12 mL, 4.3×10⁻³ mmol) were combined in the Ar_((g))purged 3-necked round bottom flask. After stirring the mixture for 20min, α-propargyl-δ-valerolactone (pvl, 0.35 g, 2.5 mmol) andδ-valerolactone (vl, 1.1 g, 10.0 mmol) were added. The reaction vesselstirred at 105° C. for 48 h. Residual monomer and catalyst were removedby precipitating the polymer into cold diethyl ether to give a goldenbrown polymer. Yield: 1.18 g (81.4%). M_(w)=3000 Da, PDI=1.18. ¹H NMR(300 MHz, CDCl₃/TMS): δ 4.10 (m, —CH₂—O—), 3.64 (m, CH₃CH₂O—), 2.59 (m,pvl, HC≡CCH₂CH—), 2.35 (m, vl, —CH₂CH₂C(O)O—, pvl, HC≡CCH₂CH—,HC≡CCH₂CH—), 2.03 (m, HC≡C—), 1.68 (m, pvl & vl, —CHCH₂CH₂—), 1.25 ppm(t, CH₃CH₂O—). ¹³C NMR (400 MHz, CDCl₃): δ 173.6 (pvl, —C(O)—), 172.5(vl, —C(O)—), 78.2 (HC≡C—), 71.3, 68.4, 63.8, 36.7, 33.6, 29.7, 28.3,24.6, 21.5, 19.0, 16.7 ppm.

91. Click Reaction Conditions for Nanoparticle Formation UtilizingPolyoxyethylene bis(azide).

Poly(pvl-vl) (10 mg, M_(w)=3000 Da, PDI=1.18) was added to a vial, whichwas then sealed and purged with argon. Polyoxyethylene bis(azide) (58.7mg, 1.2×10⁻² mmol) dissolved in anhydrous dimethylformamide (0.5 mL) andcopper (I) bromide (23.4 μL, 3.5×10⁻² M solution in DMF) were added. Thereaction mixture stirred for 24 h at room temperature. Residual azideand copper bromide were removed by dialyzing with SnakeSkin® PleatedDialysis Tubing (MWCO=25,000) against 50/50 dichloromethane/methanol.Yield: 43.4 mg. DLS: D_(H)=21.9±1.9 nm. ¹H NMR (300 MHz, CDCl₃/TMS): δThe significant change is the reduction of the alkyne proton at 2.03 ppmand the appearance of signals at 3.65 and 3.40 ppm corresponding to theprotons of the PEG linker and the signal at 7.49 ppm due to the protonsfrom triazole formation as a result of cross-linking. All other aspectsof the spectrum are similar to that of poly(vl-pvl).

92. Click Reaction Conditions for Nanoparticle Formation Utilizing1,8-diazide-3,5-dioxaoctane.

Poly(pvl-vl) (40.8 mg, M_(w)=3000 Da, PDI=1.18) was added to a vial,which was then sealed and purged with argon. To the vial,1,8-diazide-3,5-dioxaoctane (19.5 mg, 9.7×10⁻² mmol) dissolved inanhydrous dimethylformamide (0.8 mL) and copper (I) bromide (115.3 μL,5.9×10⁻² mM solution in DMF) were added. The reaction mixture stirredfor 24 h at 40° C. Residual azide and copper bromide were removed bydialyzing with SnakeSkin® Pleated Dialysis Tubing (MWCO=10,000) against50/50 CH₂Cl₂/CH₃OH. Yield: 37.8 mg. DLS: D_(H)=21.9±1.9 nm. ¹H NMR (300MHz, CDCl₃/TMS): δ The significant change is the reduction of the alkyneproton at 2.03 ppm and the appearance of signals at 3.65 and 3.40 ppmcorresponding to the protons of the PEG linker and the signal at 7.49ppm due to the protons from triazole formation as a result ofcross-linking. All other aspects of the spectrum are similar to that ofpoly(pvl-vl).

93. General Procedure for Formulating Nanoparticles with TPGS-Vitamin E.

To a 150 mL beaker containing _(D)-α-tocopheryl polyethylene glycol 1000succinate (vitamin E TPGS) (0.28 g) dissolved in Lonza cell culturewater (55 mL), nanoparticles (0.0977 g) dissolved in dimethyl sulfoxide(DMSO) (0.50 mL) were added slowly with vigorous stirring. The solutionwas split equally into two 50 mL centrifuge tubes. The nanoparticleswere rinsed by applying three cycles of centrifugation (8000 rpm for 30min) and reconstituted with cell culture water. The nanoparticlesuspension was then lyophilized.

94. General Procedure for In Vitro Cytotoxicity of FormulatedNanoparticles (MTT Assay).

The cytotoxicity of the formulated nanoparticles was evaluated using anMTT assay. HeLa cells were cultured in Eagle's Minimum Essential Mediumsupplemented with 10% heat inactivated fetal bovine serum, L-glutamine,penicillin streptomycin sulfate antibiotic-antimycotic mixture andgentamicin. Cells were maintained at 37° C. with 5% CO₂ in a 95%humidity incubator. The cells were seeded in a 96-well plate in 100 μLmedia per well at a density of 10,000 cells/well and incubated for 24 h.The media was then replaced with 100 μL of phenol red freemedium-containing nanoparticles at different concentrations intriplicate and incubated for 24 h. After incubation, the nanoparticlecontaining media was removed, the cells were rinsed three times withDPBS, to avoid interference in the assays, and 100 μL of fresh phenolred free media was added, followed by 10 μL MTT solution (5 mg/mL). Thecells were incubated for 4 h, after which time the medium was carefullyremoved. To the resulting purple crystals, 100 μL DMSO was added to lysethe cells and was incubated for 10 min at 37° C. The MTT absorbance wasmeasured at 540 nm using a Synergy HT Multi-mode microplate reader (BioTek Instruments, Winooski, Vt.). Optical densities measured for wellscontaining cells that received no nanoparticle were considered torepresent 100% viability. Results are expressed as the mean±S.D. ofviable cells.

95. Encapsulation of Brimonidine in Nanoparticles.

To a 150 mL beaker containing _(D)-α-tocopherol polyethylene glycol 1000succinate (0.15 g) dissolved in Lonza cell culture water (30 mL),nanoparticles (60.5 mg) and brimonidine (6.1 mg) dissolved in dimethylsulfoxide (0.50 mL) were added slowly with vigorous stirring. Thesolution was split equally into two 50 mL centrifuge tubes. Thebrimonidine loaded nanoparticles were purified by applying three cyclesof centrifugation (8000 rpm for 30 min) and reconstituted with cellculture water. The nanoparticle suspension was then lyophilized. Theconcentration of encapsulated brimonidine was determined by NanoDrop™UV-Vis at a wavelength of 389 nm. Brimonidine standards (0.32-1.92mg/mL) were measured by UV-Vis and a calibration curve was rendered.With the calibration curve, the concentration of encapsulatedbrimonidine was determined by the absorbency of the brimonidine in thenanoparticle at 389 nm and the loading ratio was found to be 6.5%.

96. General Procedure for the Formation of Nanoparticles frompoly(vl-evl).

In a 100 mL three-necked round bottom flask equipped with stir bar,condenser and septa, 2,2′-(ethylenedioxy)bisethylamine (34.1 μL,2.32×10⁻⁴ mol), 28.7 mL CH₂Cl₂ and a solution of poly(vl-evl) (0.14 g,M_(w)=3400 Da, PDI=1.16) in 0.19 mL CH₂Cl₂ were added. The mixture washeated at 44° C. for 12 h. Residual diamine was removed by dialyzingwith SnakeSkin® Pleated Dialysis Tubing (MWCO=10,000) againstdichloromethane. DLS: D_(H)=272.3±23.3 nm. ¹H NMR (300 MHz, CDCl₃/TMS)δ: The significant change is the disappearance of the epoxide protons at2.94, 2.75 and 2.47 ppm and the appearance of signals at 3.64 and 2.97ppm corresponding to the protons neighboring the secondary amine of thePEG linker after cross-linking. All other aspects of the spectrum aresimilar to that of poly(vl-evl), as referenced in the literature.⁸

97. General Procedure for In Vitro Nanoparticle Degradation Studies.

Poly(vl-evl) nanoparticles (10 mg) were suspended in 2 mL of Dulbecco'sPhosphate Buffered Saline (pH 7.2) in 2 dram vials equipped with stirbars. The vials were sealed to avoid evaporation and the samples weremaintained at 37° C. under continuous stirring. At 48 h intervals,samples were removed and dichloromethane was added (3×4 mL) to extractremaining nanoparticles and degradation products. The extractionsolutions were concentrated via rotary evaporator and dried in vacuo.The degradation of the nanoparticles was monitored by the change inmolecular weight, as determined by static light scattering, withincubation time.

98. General Procedure for Nanoparticle Formation frompoly(vl-evl-avl-opd).

To a solution of 2,2′-(ethylenedioxy)diethylamine (23.4 μL, 0.16 mmol)in CH₂Cl₂ (98.7 mL), a solution of poly(vl-evl-avl-opd) (0.1840 g,M_(w)=3440 Da) in CH₂Cl₂ (0.64 mL) was added. The mixture was heated at44° C. for 12 h. Residual diamine was removed by dialyzing withSnakeSkin® Pleated Dialysis Tubing (MWCO=10,000) against CH₂Cl₂. Yield:0.15 g (94%). DLS: D_(H)=52.9±3.3 nm. SLS: M_(w)=147,000 Da. ¹H NMR (300MHz, CDCl₃/TMS) δ: The significant change is the disappearance of theepoxide protons at 2.94, 2.75 and 2.47 ppm and the appearance of signalsat 3.54 and 2.97 ppm corresponding to the protons neighboring thesecondary amine of the PEG linker after cross-linking. All other aspectsof the spectrum are similar to that of poly(vl-evl-avl-opd), asreferenced in the literature.⁹

99. Formulation of poly(vl-evl-avl-opd) Nanoparticles with TPGS-VitaminE.

To a 150 mL beaker containing _(D)-α-tocopheryl polyethylene glycol 1000succinate (vitamin E TPGS) (0.28 g) dissolved in Lonza cell culturewater (55 mL), nanoparticles (0.0977 g) dissolved in dimethyl sulfoxide(DMSO) (0.50 mL) were added slowly with vigorous stirring. The solutionwas split equally into two 50 mL centrifuge tubes. The nanoparticleswere rinsed by applying three cycles of centrifugation (8000 rpm for 30min) and reconstituted with cell culture water. The nanoparticlesuspension was then lyophilized.

100. In Vitro poly(vl-evl-avl-opd) Nanoparticle Degradation Studies.

TPGS formulated poly(vl-evl-avl-vl) nanoparticles (10 mg) were suspendedin 2 mL of Dulbecco's Phosphate Buffered Saline (pH 7.4) in 2 dram vialsequipped with stir bars. The vials were sealed to avoid evaporation andthe samples were maintained at 37° C. under continuous stirring. At 48 hintervals, samples were removed and dichloromethane was added (3×4 mL)to extract remaining nanoparticles and degradation products. Theextraction solutions were concentrated via rotary evaporator and driedin vacuo. The degradation of the nanoparticles was monitored by thechange in molecular weight, as determined by static light scattering,with incubation time.

101. In Vitro Cytotoxicity of Formulated poly(vl-evl-avl-opd)Nanoparticles (MTT Assay).

The cytotoxicity of TPGS formulated nanoparticles was evaluated using anMTT assay. HeLa cells were cultured in Eagle's Minimum Essential Mediumsupplemented with 10% heat inactivated fetal bovine serum, L-glutamine,penicillin streptomycin sulfate antibiotic-antimycotic mixture andgentamicin. Cells were maintained at 37° C. with 5% CO₂ in a 95%humidity incubator. The cells were seeded in a 96-well plate in 100 μLmedia per well at a density of 10,000 cells/well and incubated for 24 h.The media was then replaced with 100 μL of phenol red freemedium-containing nanoparticles at different concentrations intriplicate and incubated for 24 h. After incubation, the nanoparticlecontaining media were removed, the cells were rinsed three times withDPBS, to avoid interference in the assays, and 100 μL of fresh phenolred free media was added, followed by 10 μL MTT solution (5 mg/mL). Thecells were incubated for 4 h, after which time the medium was carefullyremoved. To the resulting purple crystals, 100 μL DMSO was added to lysethe cells and was incubated for 10 min at 37° C. The MTT absorbance wasmeasured at 540 nm using a Synergy HT Multi-mode microplate reader (BioTek Instruments, Winooski, Vt.). Optical densities measured for wellscontaining cells that received no nanoparticle were considered torepresent 100% viability. Results are expressed as the mean±S.D. ofviable cells.

102. In Vitro Release of Paclitaxel from poly(vl-evl-avl-opd)Nanoparticles.

To a 150 mL beaker containing _(D)-α-tocopherol polyethylene glycol 1000succinate (0.34 g) dissolved in Lonza cell culture water (68 mL),poly(vl-evl-avl-opd) nanoparticles (56.5 mg) and paclitaxel (8.5 mg)dissolved in dimethyl sulfoxide (0.50 mL) were added slowly withvigorous stirring. The solution was split equally into two 50 mLcentrifuge tubes. The paclitaxel loaded nanoparticles were purified byapplying three cycles of centrifugation (8000 rpm for 30 min) andreconstituted with cell culture water. The nanoparticle suspension wasthen lyophilized. The concentration of encapsulated paclitaxel wasdetermined by NanoDrop™ UV-Vis at a wavelength of 254 nm. Paclitaxelstandards (0.398-2.39 mg/mL) were measured by UV-Vis and a calibrationcurve was rendered. With the calibration curve, the concentration ofencapsulated paclitaxel was determined by the absorbency of thepaclitaxel in the nanoparticle at 254 nm and the loading ratio was foundto be 11.3%. The release of paclitaxel from the nanoparticles wasmeasured in PBS (pH 7.4) at 37° C. The paclitaxel-loaded nanoparticles(20 mg) were suspended in PBS (20 mL). At particular time intervals, thenanoparticle dispersion was centrifuged, the supernatant was removed andthe released paclitaxel was extracted from the supernatant with CH₂Cl₂.The concentration of released paclitaxel was determined by NanoDrop™UV-Vis at a wavelength of 254 nm as mentioned above.

103. General Procedures for the Synthesis of HVGGSSV Peptide.

The peptide was synthesized by solid-phase peptide synthesis usingstandard Fmoc chemistry on a Model 90 Peptide Synthesizer (AdvancedChemTech).

General Procedure: Attachment of N-Fmoc Amino Acids to Resin.

After swelling with dichloromethane (20 mL) for 20 min, H-Val-2-Cl-Trtresin (0.20 g, 1.03 mmol/g, 0.21 mmol surface amino acids) was treatedwith a solution of Fmoc-protected amino acids (4 equiv, 0.9 mmol) indimethylformamide (DMF) (6 mL). The amino acids were attached to theresin using double coupling with a solution (9 mL) consisting ofN-hydroxybenzotriazole monohydrate (HOBt) (0.9 mmol, 137.8 mg)o-(benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (0.9mmol, 0.34 g), N,N′-diisopropylethylamine (DIPEA) (1.8 mmol, 0.31 mL) in9 mL DMF. The reaction mixture was shaken for 60 min and washed with DMF(4×10 mL), methanol (4×10 mL) and DMF (4×10 mL). A 20% (v/v) piperidinein DMF solution was used to deprotect the Fmoc groups. The amino acidswere attached to the resin in the following sequence: Ser, Ser, Gly,Gly, Val, His, Asn, Gly, Gly, Gly, Cys, and Gly.

General Procedure: Cleavage from Resin.

The resin was treated with Reagent R, a solution of TFA, thioanisole,anisole, and ethanedithiol (90:5:3:2, 6 mL), for 4 h. After removal ofthe resin by filtration, the filtrate was concentrated to precipitatethe peptide with cold diethyl ether. Crude peptides were purified byRP-HPLC and lyophilized. Peptide identity was confirmed by MALDI-MS(m/z: 1086.45).

104. Attachment of HVGGSSV Peptide to Nanoparticles.

To a solution of nanoparticles (105.6 mg, 0.78 μmol) in DMSO (1 mL),HVGGSSV peptide (56 mg, 53.6 μmol) in DMSO (2 mL) was added. Thereaction mixture was heated for 72 h at 34° C. Residual peptide wasremoved by dialyzing with SnakeSkin® Pleated Dialysis Tubing(MWCO=10,000) against 50/50 THF/CH₃CN. Yield: 77 mg. DLS: D_(H)=55.3±3.6nm; original particle D_(H)=52.9±3.3 nm. SLS: M_(w)=185,000 Da; originalparticle M_(w)=147,000 Da. ¹H NMR (600 MHz, DMSO-d₆) δ: The significantchange is the reduction of the allyl protons at 5.69 and 5.00 ppm andthe appearance of signals at 0.80, 1.39, 1.65, 2.74, 3.07, 3.75, 4.40and 7.11-8.32 ppm due to the peptide. All other aspects of the spectrumare similar to that of the poly(vl-evl-avl-opd) nanoparticles.

105. Encapsulation of Paclitaxel in HVGGSSV Conjugatedpoly(vl-evl-avl-opd) Nanoparticles.

To a 150 mL beaker containing _(D)-α-tocopherol polyethylene glycol 1000succinate (0.30 g) dissolved in Lonza cell culture water (60 mL),HVGGSSV-nanoparticles (0.0681 g) and paclitaxel (10.2 mg) dissolved indimethyl sulfoxide (0.50 mL) were added slowly with vigorous stirring.The solution was split into two 50 mL centrifuge tubes. The paclitaxelloaded nanoparticles were purified by applying two cycles ofcentrifugation (8000 rpm for 30 min) and reconstituted with cell culturewater. The nanoparticle suspension was then lyophilized. The loadingratio of paclitaxel for the encapsulation was determined by NanoDrop™UV-Vis at 254 nm as mentioned above and was found to be 11%.

106. Lowering Intraocular Pressure (IOP) with Brimonidine NanoparticleInjection

Two groups of mice (N=3 each) were compared to determine the effect of asingle intravitreal injection of brimonidine-laced nanoparticle relativeto a single topical drop (eyedrop) of clinical-grade brimonidine. Foreach mouse, intraocular pressure (IOP) was acutely elevated by aninjection (1 μl) of polystyrene microbeads into the anterior chamber ofthe eye. This induces a 35-40% elevation in IOP that persists for 3-4weeks. One group had a single topical application (1 μl) of brimonidine;the other a single intravitreal injection (1 μl) of thenanoparticle-brimonidine complex. IOP was tracked using TonoPen XLmeasurements until any lowering effect was dissipated.

For the topical application group, microbead injection induced a 35%elevation in IOP from a normal reading of 14 mmHG to 19-20 mmHG, one dayafter injection (FIG. 1). This elevation persisted until day 4, whentopical brimonidine was applied; the application lowered IOP to normallevels one day later. Six days following topical administration ofbrimonidine, IOP returned to elevated levels.

For the nanoparticle group, microbead injection again induced a 40%elevation in IOP one day after injection, from a normal baseline of 15mmHG to 21 mmHG (FIG. 2). This elevation persisted until day 4, when asingle intravitreal injection (1 μl) of the nanoparticle-brimonidinecomplex was applied. The nanoparticle complex actually lowered IOP belownormal levels to 11.5 mmHG one day later; this depression wassignificant (p<0.01). IOP remained below or at baseline for 6 days. Forthis period, IOP was indistinguishable from pre-microbead/baselinelevels (p=0.43). This is dramatically different than topicalapplication, which returned to elevated IOP during the same period. Forthe nanoparticle group, IOP returned to elevated levels by day 18.Control groups for both experiments demonstrated continuously elevatedIOP due to microbead injection for the duration.

107. Measuring Retinal Diffusion after Nanoparticle Injection

For glaucoma, there is no FDA approved neuroprotective therapy forpreventing or treating retinal and optic nerve degeneration. Allavailable drugs have as their action IOP lowering. Thus, a secondary useof the nanoparticle delivery system would be to expose the retina andoptic nerve to a slow-release of directly neuroprotective compounds,such as memantine or brimonidine, which is known to have secondaryneuronal actions independent of IOP lowering. For macular degeneration,the best available practice is a monthly or biweekly intravitrealinjection of antiangiogenic compounds, such as LUCENTIS® (ramibizumab)or AVASTIN® (bevaizumab). Again the nanoparticle delivery system couldameliorate the need for such frequent injections.

To determine how much of the retina could be stained over time with acommon neuronal dye (DiO) interlaced into the nanoparticle after asingle intravitreal injection, the area of the retina covered by DiOreleased from the nanoparticle complex was measured as a function oftime after the injection (N=2-3 mice for each time). Deposition of DiOwas defined very conservatively, as that portion of the retina containedDiO signal intensity of 100% contrast compared to background. The areaof the retina represented by DiO label was compared to the total surfacearea of the retina. Retinas were examined at 3 days, 1 week, 2 weeks,and 4 weeks post-nanoparticle injection.

Over a 4 week period, DiO deposition appeared to increase on the retinalsurface by about 15% compared to the initial measurement at 3 days; thiswas not significant (p=0.50) (FIG. 53). In between, deposition wasstatistically constant compared to the initial measurement as well. Thisindicates that retinal uptake of the DiO is fairly consistent andmatched to its slow release from the nanoparticle complex. Thus, retinalexposure to a released drug would be constant in between nanoparticleinjections. Additionally, the ability of nanoparticles to pas throughthe inner limiting membrane and deliver DiO to ganglion cells wasmeasured at 3 days, 1 week, and 2 weeks following injection. Micrographsshow that deposition was observed in ganglion cells and maintained overthe observatory period (FIG. 54).

108. Preparation of Nanoparticles from Linear Polymer Precursor

a. Formation of 50 nm Nanoparticles

To a 100-mL round bottom flask equipped with a stir bar, poly(vl-evl)(0.1001 g, M_(w)=2350 Da, 7% cross-linking) and 20.2 mL CH₂Cl₂ wereadded, followed by 2,2′-(ethylenedioxy)diethylamine (9.6 μL, 6.55×10⁵mol). The mixture was heated at reflux at 44° C. for 12 h and promptlytransferred to SnakeSkin® Pleated Dialysis Tubing (MWCO=10,000) anddialyzed against dichloromethane to remove residual diamine. ¹H NMR (400MHz), CDCl₃/TMS, ppm) δ: The significant change, proving conversion fromthe linear polymer to the nanoparticle, is the disappearance of epoxideprotons at 2.96, 2.75, and 2.47 ppm and the appearance of signals at 3.5ppm and 2.9 ppm due to the protons near the secondary amine of the PEGlinker.

b. Formation of 400 nm Nanoparticles

To a 200-mL round bottom flask equipped with a stir bar, poly(vl-evl)(0.1210 g, M_(w)=2325 Da, 13% cross-linking) and 45.1 mL CH₂Cl₂ wereadded, followed by 2,2′-(ethylenedioxy)diethylamine (75.1 μL, 5.13×10⁻⁴mol). The mixture was heated at reflux at 44° C. for 12 h and promptlytransferred to SnakeSkin® Pleated Dialysis Tubing (MWCO=10,000) anddialyzed against dichloromethane to remove residual diamine. ¹H NMR (400MHz), CDCl₃/TMS, ppm) δ: The significant change is the disappearance ofepoxide protons at 2.93, 2.76, and 2.47 ppm and the appearance ofsignals at 3.5 and 2.9 ppm, correlating to the protons of the PEGlinker.

c. Formation of 700 nm Nanoparticles

To a 200-mL round bottom flask equipped with a stir bar,poly(vl-evl-avl) (0.1057 g, M_(w)=7200 Da, 15% cross-linking) and 46.4mL CH₂Cl₂ were added, followed by 2,2′-(ethylenedioxy)diethylamine (82.5μL, 5.64×10⁻⁴ mol). The mixture was heated at reflux at 44° C. for 12 hand promptly transferred to SnakeSkin® Pleated Dialysis Tubing(MWCO=10,000) and dialyzed against dichloromethane to remove residualdiamine. ¹H NMR (400 MHz), CDCl₃/TMS, ppm) δ: The significant change isthe disappearance of epoxide protons at 2.94, 2.75, and 2.48 ppm and theappearance of signals at 3.5 and 2.9 ppm, correlating to the protons ofthe PEG linker.

d. Formation of 700 nm Nanoparticles

To a 200-mL round bottom flask equipped with a stir bar,poly(vl-evl-avl) (0.1001 g, M_(w)=7200 Da, 15% cross-linking) and 43.9mL CH₂Cl₂ were added, followed by 2,2′-(ethylenedioxy)diethylamine (39.1μL, 2.67×10−4 mol) and 1,8-diaminooctane (38.5 mg, 2.67×10⁻⁴ mol). Themixture was heated at reflux at 44° C. for 12 h and transferred toSnakeSkin® Pleated Dialysis Tubing (MWCO=10,000) and dialyzed againstdichloromethane to remove residual diamines. ¹H NMR (400 MHz),CDCl₃/TMS, ppm) δ: The significant change, confirming incorporation of1,8-diaminooctane, is the appearance of a signal at 1.32 ppmcorresponding to the protons between the secondary amines of thecross-linker. The spectrum shows otherwise similar shifts as theparticles of 50 and 400 nm.

109. General Procedure for Encapsulation of NP

The 700 nm nanoparticle with 15% cross-linking (100% PEG linker) (16.7mg) and bimatoprost(7-[3,5-dihydroxy-2-(3-hydroxy-5-phenyl-pent-1-enyl)-cyclopentyl]-N-ethyl-hept-5-enamide;a prostaglandin analog/prodrug used topically to control the progressionof glaucoma and in the management of ocular hypertension; 5.0 mg) wereaccurately weighed together into a vial. The two solids were dissolvedin a minimal amount of DMSO (150 μL) and added dropwise to a vigorouslystirring solution of water (8.3 mL) and vitamin E (0.125 g). Thesolution turned cloudy and was immediately centrifuged at 8500 rpm for20 min. The supernatant was carefully removed, fresh water was added andthe pellet disturbed to ensure thorough washing of the drug-loadedparticles. The centrifugation wash was repeated for a total of threewashes. Finally, the particles were frozen and lyophilized to yield thedrug-loaded particles as a light and fluffy white solid with 29.4%bimatoprost encapsulated.

The 700 nm nanoparticle with the 50:50 mixture of amorphous andcrystalline cross-linkers encapsulated 25.4% bimatoprost. The 400 nmnanoparticle encapsulated 22.4% bimatoprost, and the 50 nm nanoparticleencapsulated 1.3% travatan (i.e., Travoprost, propan-2-yl7-[3,5-dihydroxy-2-[3-hydroxy-4-[3-(trifluoromethyl)phenoxy]-but-1-enyl]-cyclopentyl]hept-5-enoate;topical medication used for controlling the progression of glaucoma orocular hypertension, by reducing intraocular pressure), and the other 50nm nanoparticle encapsulated 3.3% brimonidine(5-Bromo-N-(4,5-dihydro-1H-imidazol-2-yl)quinoxalin-6-amine; used totreat open-angle glaucoma or ocular hypertension).

110. Nanodrop to Determine % Drug Loading

About 0.4 mg of drug-loaded nanoparticles were weighed and dissolved in50 uL DMSO. 2 uL of sample solution was pipetted onto the pedestal of aUV-VIS spectrometer (NanoDrop) and the absorbance measured at 262 nm. Acalibration curve between concentration of drug and absorbance was madeusing a spread of samples with known concentrations of drug. Using thecalibration curve, the amount of drug within the nanoparticle could bequantified and reported as a weight percent.

111. General Procedure for Mouse Study Preparation

The nanoparticle (1.20 mg, 700 nm amorphous) was accurately weighed intoan eppendorff tube and dispersed in PBS (75 μL) for an over-allconcentration of 16 mg/mL, or 3.6 mg/mL bimatoprost.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

What is claimed is:
 1. A composition comprising a crosslinked degradable polyester particle including polymer backbones comprising δ-valerolactone monomer residues, the crosslinked degradable polyester particle comprising one or more crosslinks comprising a structure selected from: (a)

wherein Y is O, S, or N-R, wherein R is C1-C4 alkyl; (b)

and (c)

wherein L is a divalent alkyl chain or alkyloxyalkyl chain.
 2. The composition of claim 1, further comprising: at least one of a biologically active agent, a pharmaceutically active agent, or an imaging agent.
 3. The composition of claim 2, wherein the composition is produced by a nucleophilic substitution reaction between a nucleophile including at least one of the biologically active agent, the pharmaceutically active agent, or the imaging agent and the polymer backbones.
 4. The composition of claim 3, wherein at least one of the biologically active agent, the pharmaceutically active agent, or the imaging agent are attached to an optionally substituted organic radical comprising 1 to 24 carbon atoms.
 5. The composition of claim 4, wherein the biologically active agent, the pharmaceutically active agent, or the imaging agent is covalently bonded to the optionally substituted organic radical comprising 1 to 24 carbon atoms.
 6. The composition of claim 4, wherein the optionally substituted organic radical comprising 1 to 24 carbon atoms includes substituted or unsubstituted monovalent organic radicals selected from the group consisting of ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, decyl, dodecyl, hexadecyl, higher cyclic or acyclic alkyl, and alkoxylene.
 7. The composition of claim 2, wherein the at least one of a biologically active agent, a pharmaceutically active agent, or an imaging agent are encapsulated within the a crosslinked degradable polyester particle.
 8. The composition of claim 1, wherein the crosslinked degradable polyester particle is a copolymer and/or a nanoparticle. 